Methods of treating cancer

ABSTRACT

Provided are methods of treating cancer (e.g., non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), HER2-positive gastric/gastroesophageal junction (GEJ) cancer, de novo or transformed diffuse large B cell lymphoma (DLBCL), or indolent lymphoma) in an individual that comprise administering to the individual (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-cancer antibody (e.g., an anti-PD1 antibody, anti-HER2 antibody, or an anti-CD20 antibody). Also provided are related kits pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication No. 62/855,821, filed May 31, 2019 and U.S. ProvisionalApplication No. 63/022,187, filed May 8, 2020, the contents of each ofwhich are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 757972001000SEQLIST.TXT,date recorded: May 26, 2020, size: 333 KB).

FIELD OF THE INVENTION

The present invention relates to methods of treating cancer thatcomprise administering a polypeptide (e.g. a fusion polypeptide) thatcomprises a SIRPα D1 domain variant and an Fc domain variant inconjunction with a therapeutic antibody.

BACKGROUND

Many cancers have a poor prognosis, even when treated with availabletherapeutics. For example, non-small cell lung cancer (NSCLC) patientswith metastatic disease receiving PD-1 and PD-L1 checkpoint inhibitorswho have failed prior platinum based therapies have a median overallsurvival rate of approximately one year (Garon et al., New Engl J Med(2015) 372:2018-28; Herbst et al., Lancet (2016) 387:1540-50;Fehrenbacher et al., Lancet (2016) 387(10030):1837-46), and over halfNSCLC patients with advanced stage disease have an overall 5-yearsurvival rate of 17.7% (U.S. Cancer Statistics Working Group, availableat the web site www(dot)cdc(dot)gov/uscs). Similarly, the overall 5-yearsurvival rate for gastric cancer patients in the United States is 30.4%(U.S. Cancer Statistics Working Group). In patients with relapsedindolent lymphomas, subsequent relapses usually occur with increasinglyaggressive histologies and a transformation risk of 30% by 10 years inone series (Montoto et al., J Clin Oncol (2007) 25(17):2426-33).Further, for patients with recurrent aggressive histologies, cure israre, and novel salvage regimens are needed (Larouche et al., J ClinOncol (2010) 28(12):2094-100). CD20-positive non-Hodgkin lymphoma (NHL)as the 10th most common cancer globally, is also the 10th leading causeof cancer death, accounting for 199,670 deaths per year, worldwide(World Health Organization 2016(a), available at the websiteglobocan(dot)iarc(dot)fr/Pages/fact_sheets_cancer(dot)aspx). There areestimated to be over 35,000 people living in the US with metastaticHNSCC, with over 50,000 newly incident cases at all stages diagnosed in2019. Five-year survival is 84% for patients diagnosed with localizeddisease but decreases to only 39% for those diagnosed with metastaticdisease. There is a need in the art for new treatments to provideadditional therapeutic options and improve outcomes for such patients.

Tumor cells manipulate the myeloid compartment to evade the anti-tumorhost immune response (Gabrilovich et al., Nat Rev Immunol (2012)12(4):253-68). For example, while CD47 expressed on the surface ofnormal cells binds SIRPα on macrophages and provides a “don't eat me”signal, tumor cells have also been found to overexpress CD47 to evadethe macrophage component of immune surveillance (Oldenborg, ISRN Hematol(2013) 614619).

Macrophage-mediated destruction of cancer cells requires both thedisruption of “don't eat me” signals (e.g., CD47-SIRPα) and theactivation of “eat me” signals. Neither component alone is sufficient totrigger maximal phagocytic reaction against tumor cells. As describedabove, CD47 provides a fundamental “don't eat me” signal through itsinteraction with SIRPα on macrophages. The pro-phagocytic “eat me”signal can be provided to the same macrophages by binding to theiractivating Fc gamma receptors. For example, the pro-phagocytic “eat me”signal can be provided by binding of anti-tumor antibodies to Fcreceptors on macrophages.

All references cited herein, including patent applications, patentpublications, and UniProtKB/Swiss-Prot Accession numbers are hereinincorporated by reference in their entirety, as if each individualreference were specifically and individually indicated to beincorporated by reference.

SUMMARY OF THE INVENTION

Provided is a method of treating non-small cell lung cancer (NSCLC) inan individual, comprising administering to the individual an effectiveamount of (a) a polypeptide comprising a SIRPα D1 domain variant and anFc domain variant, and (b) an anti-PD-1 antibody, wherein the SIRPα D1domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85; wherein the Fc domain variant is (i) a human IgG Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the individual is a human. In some embodiments, the NSCLC in theindividual has progressed on a prior immune checkpoint inhibitor (CPI)therapy and/or has a PD-L1 tumor proportion score (TPS) of less than50%,

Also provided is a polypeptide comprising a SIRPα D1 domain variant andan Fc domain variant for use in the manufacture of a medicament fortreating NSCLC in an individual, wherein the medicament is for use (suchas formulated for use) in combination with an anti-PD1 antibody, whereinthe SIRPα D1 domain variant comprises the amino acid sequence of SEQ IDNO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a humanIgG Fc region comprising L234A, L235A, G237A, and N297A mutations,wherein numbering is according to the EU index of Kabat; (ii) a humanIgG2 Fc region comprising A330S, P331S, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (iii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations,wherein numbering is according to the EU index of Kabat; or (iv) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, deG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the individual is a human. In some embodiments, the NSCLC in theindividual has progressed on a prior immune checkpoint inhibitor (CPI)therapy and/or has a PD-L1 tumor proportion score (TPS) of less than50%.

Also provided is a composition (such a pharmaceutical composition)comprising a polypeptide comprising a SIRPα D1 domain variant and an Fcdomain variant for use in combination with an anti-PD1 antibody fortreating NSCLC in an individual (e.g., for use in a method of treatingNSCLC in an individual), wherein the SIRPα D1 domain variant comprisesthe amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein theFc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A,G237A, and N297A mutations, wherein numbering is according to the EUindex of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, andN297A mutations, wherein numbering is according to the EU index ofKabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, and delG236 mutations, wherein numbering is according to the EUindex of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, delG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; and wherein the individual is ahuman. In some embodiments, the NSCLC in the individual has progressedon a prior immune checkpoint inhibitor (CPI) therapy and/or has a PD-L1tumor proportion score (TPS) of less than 50%.

In some embodiments, the prior CPI (immune checkpoint inhibitor therapy)comprised one or more agents selected from the group consisting of:nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, andcemiplimab. In some embodiments, the anti-PD-1 antibody blocks theinteraction between PD-1 and PD-L1. In some embodiments, the anti-PD-1antibody is pembrolizumab. In some embodiments, the pembrolizumab isadministered to the individual at a dose of 200 mg every 3 weeks (Q3W)by intravenous (IV) infusion.

Also provided is a method of treating head and neck squamous cellcarcinoma (HNSCC) in an individual, comprising administering to theindividual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-PD-1antibody, wherein the SIRPα D1 domain variant comprises the amino acidsequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domainvariant is (i) a human IgG Fc region comprising L234A, L235A, G237A, andN297A mutations, wherein numbering is according to the EU index ofKabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297Amutations, wherein numbering is according to the EU index of Kabat;(iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, anddelG236 mutations, wherein numbering is according to the EU index ofKabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, deG236, and N297A mutations, wherein numbering is according tothe EU index of Kabat; wherein the HNSCC in the individual hasprogressed while on a prior platinum therapy or after the platinumtherapy, and wherein the individual is a human.

Also provided is a polypeptide comprising a SIRPα D1 domain variant andan Fc domain variant for use in the manufacture of a medicament fortreating HNSCC in an individual, wherein the medicament is for use (suchas formulated for use) in combination with an anti-PD1 antibody, whereinthe SIRPα D1 domain variant comprises the amino acid sequence of SEQ IDNO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a humanIgG Fc region comprising L234A, L235A, G237A, and N297A mutations,wherein numbering is according to the EU index of Kabat; (ii) a humanIgG2 Fc region comprising A330S, P331S, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (iii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations,wherein numbering is according to the EU index of Kabat; or (iv) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, deG236, and N297Amutations, wherein numbering is according to the EU index of Kabat;wherein the HNSCC in the individual has progressed while on a priorplatinum therapy or after the platinum therapy, and wherein theindividual is a human.

Also provided is a composition (such a pharmaceutical composition)comprising a polypeptide comprising a SIRPα D1 domain variant and an Fcdomain variant for use in combination with an anti-PD1 antibody fortreating HNSCC in an individual (e.g., for use in a method of treatingHNSCC in an individual), wherein the SIRPα D1 domain variant comprisesthe amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein theFc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A,G237A, and N297A mutations, wherein numbering is according to the EUindex of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, andN297A mutations, wherein numbering is according to the EU index ofKabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, and delG236 mutations, wherein numbering is according to the EUindex of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, delG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; wherein the HNSCC in the individualhas progressed while on a prior platinum therapy or after the platinumtherapy, and wherein the individual is a human.

In some embodiments, the individual with HNSCC received prior therapywith an immune checkpoint inhibitor (e.g., an immune checkpointinhibitor described herein). In some embodiments, such individual isconsidered to be/referred to as “checkpoint inhibitor experienced.” Insome embodiments, the individual with HNSCC has not received priortherapy with an immune checkpoint inhibitor. In some embodiments, suchindividual is considered to be/referred to as “checkpoint inhibitornaïve.” In some embodiments, the prior platinum therapy comprised one ormore therapeutic agents selected from the group consisting of:cisplatin, carboplatin, and oxaliplatin. In some embodiments, theanti-PD-1 antibody blocks the interaction between PD-1 and PD-L1. Insome embodiments, the anti-PD-1 antibody is pembrolizumab. In someembodiments, the pembrolizumab is administered (such as formulated foradministration) to the individual at a dose of 200 mg every 3 weeks(Q3W) by intravenous (IV) infusion.

Provided is a method of treating HER2-positive gastric/gastroesophagealjunction (GEJ) cancer in an individual, comprising administering to theindividual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-HER2antibody, wherein the SIRPα D1 domain variant comprises the amino acidsequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domainvariant is (i) a human IgG Fc region comprising L234A, L235A, G237A, andN297A mutations, wherein numbering is according to the EU index ofKabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297Amutations, wherein numbering is according to the EU index of Kabat;(iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, anddelG236 mutations, wherein numbering is according to the EU index ofKabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, delG236, and N297A mutations, wherein numbering is according tothe EU index of Kabat; wherein the gastric/GEJ cancer in the individualhas progressed following a prior treatment with a fluoropyrimidine-basedtherapy and/or a prior treatment with an anti-HER2 antibody, and whereinthe individual is a human.

Also provided is a polypeptide comprising a SIRPα D1 domain variant andan Fc domain variant for use in the manufacture of a medicament fortreating HER2-positive gastric/gastroesophageal junction (GEJ) cancer inan individual, wherein the medicament is for use (such as formulated foruse) in combination with an anti-HER2 antibody, wherein the SIRPα D1domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat;wherein the gastric/GEJ cancer in the individual has progressedfollowing a prior treatment with a fluoropyrimidine-based therapy and/ora prior treatment with an anti-HER2 antibody, and wherein the individualis a human.

Also provided is a composition (such a pharmaceutical composition)comprising a polypeptide comprising a SIRPα D1 domain variant and an Fcdomain variant for use in combination with an anti-HER2 antibody fortreating HER2-positive gastric/gastroesophageal junction (GEJ) cancer inan individual (e.g., for use in a method of treating HER2-positivegastric/gastroesophageal junction (GEJ) cancer in an individual),wherein the SIRPα D1 domain variant comprises the amino acid sequence ofSEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) ahuman IgG1 Fc region comprising L234A, L235A, G237A, and N297Amutations, wherein numbering is according to the EU index of Kabat; (ii)a human IgG2 Fc region comprising A330S, P331S, and N297A mutations,wherein numbering is according to the EU index of Kabat; (iii) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236mutations, wherein numbering is according to the EU index of Kabat; or(iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations, wherein numbering is according to the EUindex of Kabat; wherein the gastric/GEJ cancer in the individual hasprogressed following a prior treatment with a fluoropyrimidine-basedtherapy and/or a prior treatment with an anti-HER2 antibody, and whereinthe individual is a human.

In some embodiments, the prior treatment with the fluoropyrimidine-basedtherapy or the prior treatment with the anti-HER2 antibody comprised oneor more therapeutic agents selected from the group consisting of:trastuzumab, pertuzumab, 5-fluorouracil, capecitabine, margetuximab, andFOLFOX. In some embodiments, the anti-HER2 antibody is trastuzumab. Insome embodiments, trastuzumab is administered (such as formulated foradministration) to the individual at an initial dose of 8 mg/kg and at adose of 6 mg/kg for each subsequent dose, and wherein trastuzumab isadministered to the individual by IV infusion every 3 weeks (Q3W).

Provided is a method of treating aggressive non-Hodgkin lymphoma (NHL)in an individual, comprising administering to the individual aneffective amount of (a) a polypeptide comprising a SIRPα D1 domainvariant and an Fc domain variant, and (b) an anti-CD20 antibody, whereinthe SIRPα D1 domain variant comprises the amino acid sequence of SEQ IDNO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a humanIgG Fc region comprising L234A, L235A, G237A, and N297A mutations,wherein numbering is according to the EU index of Kabat; (ii) a humanIgG2 Fc region comprising A330S, P331S, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (iii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations,wherein numbering is according to the EU index of Kabat; or (iv) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, deG236, and N297Amutations, wherein numbering is according to the EU index of Kabat;wherein the aggressive NHL in the individual is relapsed and/orrefractory to a prior treatment for aggressive NHL and there is noavailable curative therapy, and wherein the individual is a human.

Also provided is a polypeptide comprising a SIRPα D1 domain variant andan Fc domain variant for use in the manufacture of a medicament fortreating aggressive non-Hodgkin lymphoma (NHL) in an individual, whereinthe medicament is for use (such as formulated for use) in combinationwith an anti-CD20 antibody, wherein the SIRPα D1 domain variantcomprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85;wherein the Fc domain variant is (i) a human IgG1 Fc region comprisingL234A, L235A, G237A, and N297A mutations, wherein numbering is accordingto the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S,P331S, and N297A mutations, wherein numbering is according to the EUindex of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, and deG236 mutations, wherein numbering is according tothe EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P,E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; wherein the aggressive NHL in theindividual is relapsed and/or refractory to a prior treatment foraggressive NHL and there is no available curative therapy, and whereinthe individual is a human.

Also provided is a composition (such a pharmaceutical composition)comprising a polypeptide comprising a SIRPα D1 domain variant and an Fcdomain variant for use in combination with an anti-CD20 antibody fortreating aggressive non-Hodgkin lymphoma (NHL) in an individual (e.g.,for use in a method of treating aggressive NHL), wherein the SIRPα D1domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85; wherein the Fc domain variant is (i) a human IgG Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and deG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat;wherein the aggressive NHL in the individual is relapsed and/orrefractory to a prior treatment for aggressive NHL and there is noavailable curative therapy, and wherein the individual is a human.

In some embodiments, the aggressive NHL is diffuse large B-cell lymphoma(DLBCL), e.g., de novo DLBCL or transformed DLBCL. In some embodiments,the prior treatment(s) for aggressive NHL comprised rituximab,cyclophosphamide, doxorubicin, vincristine, gemcitabine, lenalidomide,prednisone, prednisolone, etoposide, procarbazine, epirubicin,bendamustine, cisplatin, oxaliplatin, cytarabine, ifosfaide,carboplatin, dexamethasone, mesna, carmustine nielphalan, solumedrol,methyl-glyoxal-bis(guanylhydrazone), thiotepa, methotrexate, ibrutinib,obinituzumab, tisagenlecleucel, axicabtagene, brentuximab vedotin, andcombinations thereof. In some embodiments, the anti-CD20 antibody isrituximab. In some embodiments, the rituximab is administered (such asformulated for administration) to the individual at a dose of 375 mg/m²by IV infusion, wherein rituximab is administered (such as formulatedfor administration) to the individual once per week for four weeks andonce per month thereafter.

Provided is a method of treating indolent lymphoma in an individual,comprising administering to the individual an effective amount of (a) apolypeptide comprising a SIRPα D1 domain variant and an Fe domainvariant, and (b) an anti-CD20 antibody, wherein the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat;wherein the indolent lymphoma in the individual is relapsed and/orrefractory to a prior treatment for indolent lymphoma, and wherein theindividual is a human.

Also provided is a polypeptide comprising a SIRPα D1 domain variant andan Fc domain variant for use in the manufacture of a medicament fortreating indolent lymphoma in an individual, wherein the medicament isfor use (such as formulated for use) in combination with an anti-CD20antibody, wherein the SIRPα D1 domain variant comprises the amino acidsequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domainvariant is (i) a human IgG Fc region comprising L234A, L235A, G237A, andN297A mutations, wherein numbering is according to the EU index ofKabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297Amutations, wherein numbering is according to the EU index of Kabat;(iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, anddelG236 mutations, wherein numbering is according to the EU index ofKabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, deG236, and N297A mutations, wherein numbering is according tothe EU index of Kabat; wherein the indolent lymphoma in the individualis relapsed and/or refractory to a prior treatment for indolentlymphoma, and wherein the individual is a human.

Also provided is a composition (such a pharmaceutical composition)comprising a polypeptide comprising a SIRPα D1 domain variant and an Fcdomain variant for use in combination with an anti-CD20 antibody fortreating indolent lymphoma in an individual (e.g., for use in a methodof treating indolent lymphoma in an individual), wherein the SIRPα D1domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85; wherein the Fc domain variant is (i) a human IgG Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat;wherein the indolent lymphoma in the individual is relapsed and/orrefractory to a prior treatment for indolent lymphoma, and wherein theindividual is a human.

In some embodiments, the indolent lymphoma is an indolent non-Hodgkinlymphoma (NHL). In some embodiments, the indolent NHL is a marginal zonelymphoma or a follicular lymphoma. In some embodiments, the priortreatment for indolent lymphoma comprised rituximab, cyclophosphamide,doxorubicin, vincristine, gemcitabine, lenalidomide, prednisone,prednisolone, etoposide, procarbazine, epirubicin, bendamustine,cisplatin, oxaliplatin, cytarabine, ifosfamide, carboplatin,dexamethasone, mesna, carmustine, melphalan, solumedrol,methyl-glyoxal-bis(guanylhydrazone), thiotepa, methotrexate, ibrutinib,obinituzumab, tisagenlecleucel, axicabtagene, brentuximab vedotin,fludarabine mitoxantrone, everolimus, bortezomib, navitoclax, andcombinations thereof. In some embodiments, the anti-CD20 antibody isrituximab. In some embodiments, the rituximab is administered (such asformulated for administration) to the individual at a dose of 375 mg/m²by IV infusion, wherein rituximab is administered (such as formulatedfor administration) to the individual once per week for four weeks andonce per month thereafter. In some embodiments, the polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant (such asthe medicament manufactured using such polypeptide or a pharmaceuticalcomposition comprising such polypeptide) is administered (such asformulated for administration) to the individual at a dose of 10 mg/kgor 15 mg/kg once per week (QW), e.g., by IV infusion.

In some embodiments of any of the methods herein, the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 85. In someembodiments, the SIRPα D1 domain variant comprises the amino acidsequence of SEQ ID NOL: 81. In some embodiments, the Fc domain variantis a human IgG1 Fc region comprising L234A, L235A, G237A, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the Fc domain variant comprises the amino acidsequence of SEQ ID NO: 91. In some embodiments, the polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant comprisesthe amino acid sequence of SEQ ID NO: 136. In some embodiments, thepolypeptide comprising a SIRPα D domain variant and an Fc domain variantcomprises the amino acid sequence of SEQ ID NO: 135. In someembodiments, the polypeptide comprising a SIRPα D1 domain variant and anFc domain variant forms a homodimer.

In some embodiments, the polypeptide comprising a SIRPα D1 domainvariant and an Fe domain variant (such as the medicament manufacturedusing such polypeptide or a pharmaceutical composition comprising suchpolypeptide) is administered (such as formulated for administration) tothe individual at a dose of 10 mg/kg once per week (QW). In someembodiments, the polypeptide comprising a SIRPα D1 domain variant and anFc domain variant (or the medicament manufactured therefrom or thepharmaceutical composition comprising such polypeptide) is administered(such as formulated for administration) to the individual by IVinfusion.

Also provided is a kit comprising a polypeptide comprising a SIRPα D1domain variant and an Fc domain variant (such as the medicamentmanufactured using such polypeptide or a pharmaceutical compositioncomprising such polypeptide), for use in combination with pembrolizumabfor treating non-small cell lung cancer (NSCLC) in an individual (e.g.,human individual) in need thereof, according to a method describedherein. In some embodiments, the SIRPα D1 domain variant comprises theamino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domainvariant is (i) a human IgG Fc region comprising L234A, L235A, G237A, andN297A mutations, wherein numbering is according to the EU index ofKabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297Amutations, wherein numbering is according to the EU index of Kabat;(iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, anddeG236 mutations, wherein numbering is according to the EU index ofKabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, delG236, and N297A mutations, wherein numbering is according tothe EU index of Kabat. In some embodiments, the NSCLC in the individualhas progressed on a prior immune checkpoint inhibitor (CPI) therapyand/or has a PD-L1 tumor proportion score (TPS) of less than 50%. Insome embodiments, the kit further comprises instructions foradministering pembrolizumab at a dose of 200 mg every 3 weeks (Q3W) byIV infusion. In some embodiments, the kit further comprises instructionsfor administering the polypeptide (e.g. fusion polypeptide) at a dose of10 mg/kg once a week, e.g., by IV infusion.

Also provided is a kit comprising a polypeptide comprising a SIRPα D1domain variant and an Fc domain variant (such as the medicamentmanufactured using such polypeptide or a pharmaceutical compositioncomprising such polypeptide) for use in combination with pembrolizumabfor treating head and neck squamous cell carcinoma (HNSCC) in anindividual (e.g., a human individual) in need thereof, according to amethod described herein. In some embodiments, the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:85; the Fc domain variant is (i) a human IgG Fc region comprising L234A,L235A, G237A, and N297A mutations, wherein numbering is according to theEU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S,and N297A mutations, wherein numbering is according to the EU index ofKabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, and delG236 mutations, wherein numbering is according to the EUindex of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, delG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; and the HNSCC in the individual hasprogressed while on a prior platinum therapy or after the platinumtherapy. In some embodiments, the individual received prior therapy withan immune checkpoint inhibitor. In some embodiments, the individual hasnot received prior therapy with an immune checkpoint inhibitor. In someembodiments, the kit further comprises instructions for administeringpembrolizumab at a dose of 200 mg every 3 weeks (Q3W) by IV infusion. Insome embodiments, the kit further comprises instructions foradministering the polypeptide (e.g. fusion polypeptide) at a dose of 10mg/kg once a week, e.g., by IV infusion.

Also provided herein is a kit comprising a polypeptide comprising aSIRPα D1 domain variant and an Fc domain variant in a pharmaceuticallyacceptable carrier for use in combination with trastuzumab for treatingHER2-positive gastric/gastroesophageal junction (GEJ) cancer in anindividual (e.g., a human individual) in need thereof, according to amethod described herein. In some embodiments, the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:85; the Fc domain variant is (i) a human IgG Fc region comprising L234A,L235A, G237A, and N297A mutations, wherein numbering is according to theEU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S,and N297A mutations, wherein numbering is according to the EU index ofKabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, and delG236 mutations, wherein numbering is according to the EUindex of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, deG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; and the HER2-positive gastric/GEJcancer in the individual has progressed following a priorfluoropyrimidine-based therapy or a prior treatment with an anti-HER2antibody. In some embodiments, the kit further comprises instructionsfor administering trastuzumab at an initial dose of 8 mg/kg and at adose of 6 mg/kg for each subsequent dose, and wherein trastuzumab isadministered to the individual by IV infusion every 3 weeks (Q3W). Insome embodiments, the kit further comprises instructions foradministering the polypeptide (e.g. fusion polypeptide) at a dose of 10mg/kg once a week, e.g., by IV infusion.

Provided is a kit comprising a polypeptide comprising a SIRPα D1 domainvariant and an Fe domain variant in a pharmaceutically acceptablecarrier for use in combination with rituximab for treating aggressivenon-Hodgkin lymphoma (NHL) in an individual (e.g., a human individual)in need thereof, according to a method described herein. In someembodiments, the SIRPα D1 domain variant comprises the amino acidsequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i)a human IgG Fc region comprising L234A, L235A, G237A, and N297Amutations, wherein numbering is according to the EU index of Kabat; (ii)a human IgG2 Fc region comprising A330S, P331S, and N297A mutations,wherein numbering is according to the EU index of Kabat; (iii) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, and deG236mutations, wherein numbering is according to the EU index of Kabat; or(iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations, wherein numbering is according to the EUindex of Kabat; and the aggressive NHL in the individual is relapsedand/or refractory to a prior treatment for aggressive NHL and there isno available curative therapy. In some embodiments, the aggressive NHLis a diffuse large B-cell lymphoma (DLBCL), e.g., a de novo DLBCL or atransformed DLBCL. In some embodiments, the aggressive NHL is a mantlecell lymphoma. In some embodiments, the kit further comprisesinstructions for administering rituximab at a dose of 375 mg/m² by IVinfusion, wherein rituximab is administered to the individual once perweek for four weeks and once per month thereafter. In some embodiments,the kit further comprises instructions for administering the polypeptide(e.g. fusion polypeptide) at a dose of 10 mg/kg or 15 mg/kg once a week,e.g., by IV infusion.

Also provided is a kit comprising a polypeptide comprising a SIRPα D1domain variant and an Fc domain variant in a pharmaceutically acceptablecarrier, for use in combination with rituximab for treating indolentlymphoma in an individual (e.g., a human individual) in need thereof,according to a method described herein. In some embodiments, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 orSEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and deG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andthe indolent lymphoma in the individual is relapsed or refractory to aprior treatment for indolent lymphoma. In some embodiments, the indolentlymphoma is an indolent non-Hodgkin lymphoma (NHL). In some embodiments,the indolent NHL is a Marginal zone lymphoma or a follicular lymphoma.In some embodiments, the kit further comprises instructions foradministering rituximab at a dose of 375 mg/m² by IV infusion, whereinrituximab is administered to the individual once per week for four weeksand once per month thereafter. In some embodiments, the kit furthercomprises instructions for administering the polypeptide (e.g. fusionpolypeptide) at a dose of 10 mg/kg or 15 mg/kg once a week, e.g., by IVinfusion.

In some embodiments of any of the kits, the SIRPα D1 domain variantcomprises the amino acid sequence of SEQ ID NO: 85. In some embodiments,the SIRPα D1 domain variant comprises the amino acid sequence of SEQ IDNOL: 81. In some embodiments, the Fc domain variant is a human IgG Fcregion comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat. In some embodiments,the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the polypeptide comprising a SIRPα D1 domainvariant and an Fc domain variant comprises the amino acid sequence ofSEQ ID NO: 136. In some embodiments, the polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant comprises the amino acidsequence of SEQ ID NO: 135. In some embodiments, the polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant forms ahomodimer. In some embodiments, further comprising instructions foradministering the polypeptide comprising a SIRPα D1 domain variant andan Fc domain variant (or the medicament manufactured therefrom or thepharmaceutical composition comprising such polypeptide) to theindividual at a dose of 10 mg/kg once per week (QW). In someembodiments, the kit comprises instructions for administering thepolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant (or the medicament manufactured therefrom or the pharmaceuticalcomposition comprising such polypeptide) to the individual by IVinfusion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depicts Drug A is a monomer (FIG. 1A), and as a homodimer(FIG. 1B). The SIRPα D1 domain variant selectively binds CD47 withpicomolar binding affinity to block its interactions with SIRPα. Themolecular weight of Drug A is half the size of a typical antibody,allowing twice the molar concentration to be delivered to tumors. The Fcdomain is modified to eliminate binding to Fc gamma receptors tominimize toxicity. Drug A exhibits antibody-like pharmacokinetics.

FIG. 2 provides a schematic of the clinical study described in Example1.

FIGS. 3A-3C show the clinical activity of Drug A+trastuzumab in the HER2positive gastric/GEJ cancer expansion cohort. FIG. 3A provides thechange in tumor size, shown as percent from baseline. ORR=overallresponse rate; DCR=disease control rate; mPFS=median progression-free;CI=confidence interval. The changes in tumor sizes shown as percent frombaseline for each patient over the course of the study are provided inFIG. 3B. For FIGS. 3A-3B: patients who received at least one dose ofDrug A in the expansion phase, had a baseline assessment, and at leastone post-baseline disease assessment are included; the top dashed lineindicates the threshold for objective progression; the bottom dashedline indicates the threshold for overall response. The duration oftreatment for each enrolled patient who received at least one dose ofDrug A in the expansion phase is provided in FIG. 3C. Tumors wereassessed according to the Response Evaluation Criteria in Solid Tumors(RECIST) version 1.1 (E. A. Eisenhauer, et al., European Journal ofCancer 45 (2009) 228-247.)

FIGS. 4A-4C show the clinical activity of Drug A+pembrolizumab in theHNSCC expansion cohort. FIG. 4A provides the change in tumor size, shownas percent from baseline. ORR=overall response rate; DCR=disease controlrate; mPFS=median progression-free survival; CI=confidence interval;CP=immune checkpoint therapy; CPS=combination positive score for PD-L1staining. The changes in tumor sizes shown as percent from baseline foreach patient over the course of the study are provided in FIG. 4B. ForFIGS. 4A-4B: patients who received at least one dose of Drug A in theexpansion phase, had a baseline assessment, and at least onepost-baseline disease assessment are included; the top dashed lineindicates the threshold for objective progression; the bottom dashedline indicates the threshold for overall response. The duration oftreatment for each enrolled patient who received at least one dose ofDrug A in the expansion phase is provided in FIG. 4C. Tumors wereassessed according to the Response Evaluation Criteria in Solid Tumors(RECIST) version 1.1 (E. A. Eisenhauer, et al., European Journal ofCancer 45 (2009) 228-247.)

FIGS. 5A-5C show the clinical activity of Drug A+pembrolizumab in theNSCLC expansion cohort. FIG. 5A provides the change in tumor size, shownas percent from baseline. DCR=disease control rate; mPFS=medianprogression-free survival; CI=confidence interval; TPS=tumor proportionscore for PD-L1 staining. The changes in tumor sizes shown as percentfrom baseline for each patient over the course of the study are providedin FIG. 5B. For FIGS. 5A-5B: patients who received at least one dose ofDrug A in the expansion phase, had a baseline assessment, and at leastone post-baseline disease assessment are included; the top dashed lineindicates the threshold for objective progression; the bottom dashedline indicates the threshold for partial response. The duration oftreatment for each enrolled patient who received at least one dose ofDrug A in the expansion phase is provided in FIG. 5C. Tumors wereassessed according to the Response Evaluation Criteria in Solid Tumors(RECIST) version 1.1 (E. A. Eisenhauer, et al., European Journal ofCancer 45 (2009) 228-247.)

FIGS. 6A-6B provide pharmacokinetic and CD47 target occupancy parametersof Drug A administered in combination with pembrolizumab or trastuzumabover the course of the study. As shown in FIG. 6A, Drug A PK (Drug Aserum concentration) were within predicted 95% intervals based on anestablished population PK model. The steady-state half-life of Drug A(10 mg/kg QW) was predicted to be approximately 16 days. Drug A PK timepoints included intensive sampling in cycles 1 and 3, and pre-dose andend-of-infusion only in every cycle thereafter. FIG. 6B shows that nearcomplete CD47 target occupancy is maintained in CD4+ T cells thatexpress CD47 throughout the Drug A dosing interval when Drug A wasadministered in combination with pembrolizumab or trastuzumab. TO=targetoccupancy.

FIGS. 7A-7C show the anti-tumor response in patients treated with Drug Ain combination with pembrolizumab or trastuzumab. The percent positiveintra-tumoral CD68+ (left), CD163+ (middle), and CD8+ (right) cells inpaired biopsies before and after treatment from 5 NSCLC patients treatedwith Drug A+pembrolizumab, 6 HNSCC patients treated with DrugA+pembrolizumab and 1 gastric cancer patient treated with DrugA+trastuzumab are provided in FIG. 7A. Tumor-associated macrophages andinfiltrating lymphocytes were increased after treatment. Images from abiopsy of an NSCLC PD-L1(−) patient treated with Drug A+pembrolizumabshowing staining for CD68 and CD8 before and during treatment (C3=Cycle3) are provided in FIG. 7B. Images from a biopsy of an HNSCC PD-L1(+)patient treated with Drug A+pembrolizumab showing staining for CD68 andCD8 before and during treatment (Cycle 3) are provided in FIG. 7C. InFIGS. 7B-7C: the graph to the right shows the changes in tumor sizes aspercent from baseline for each patient over the course of the study; thetop dashed line indicates the threshold for objective progression; thebottom dashed line indicates the threshold for partial response. CD68and CD163 markers indicate macrophages; the CD8 marker indicates Tlymphocytes.

FIG. 8 provides a schematic depiction of a proposed mechanism for theanti-tumor activity of Drug A when administered in combination withanti-cancer therapeutic antibodies such as pembrolizumab, trastuzumab,or rituximab.

FIG. 9A provides a schematic showing the clinical activity of Drug A incombination with pembrolizumab in response-evaluable ≥2L HNSCC patients(including both checkpoint inhibitor-naïve patients in checkpointinhibitor-experienced patients). CPS=combined positive score (i.e., thenumber of PD-L1 positive staining cells (tumor cells, lymphocytes, andmacrophages) divided by the total number of viable tumor cells,multiplied by 100).

FIG. 9B provides a schematic showing the best overall response andduration of response in ≥2L HNSCC patients treated with Drug A andpembrolizumab.

FIG. 10A provides a schematic showing the clinical activity of Drug A incombination with pembrolizumab in response-evaluable ≥2L NSCLC patients.Patient indicated with* demonstrated change greater than 80%. TPS=tumorproportion score. FIG. 10B provides a schematic showing the best overallresponse and duration of response in ≥2L NSCLC patients treated withDrug A and pembrolizumab.

FIG. 11A provides a schematic showing the clinical activity of Drug A incombination with trastuzumab in response-evaluable ≥2L HER2⁺ gastric orHER2⁺ GEJ cancer patients. One patient with clinical progression notshown. HER2 score reflects the HER2 expression level of tumor cells.FIG. 11B provides a schematic showing the best overall response andduration of response in ≥2L HER2⁺ gastric or HER2⁺ GEJ cancer patientstreated with Drug A and trastuzumab.

FIG. 12 provides a schematic showing the clinical activity of Drug A incombination with rituximab in response-evaluable patients withrelapsed/refractory non-Hodgkin lymphoma. Patients denoted with “∨”demonstrated a more than 80% increase in measurable lesion compared tobaseline. Patients denoted with “*” achieved complete response ormetabolic complete response. Patients denoted with “mPR” achievedmetabolic partial response. Two patients (1 each at 10 mg/kg and 15mg/kg Drug A) with clinical progression are not shown. One patient (10mg/kg Drug A) with metabolic CR is not represented in the plots.

FIG. 13A provides a schematic showing the best overall response andduration of response in patients relapsed/refractory non-Hodgkinlymphoma treated with 10 mg/kg Drug A and rituximab. FIG. 13B provides aschematic showing the best overall response and duration of response inpatients relapsed/refractory non-Hodgkin lymphoma treated with 15 mg/kgDrug A and rituximab. One patient with clinical progression Study Day 8as best response not shown.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated the term “about” meaning within an acceptable errorrange for the particular value should be assumed.

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. As used herein, thesingular forms “a”, “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including”, “includes”,“having”, “has”, “with”, or variants thereof are used in either thedetailed description or the claims, such terms are intended to beinclusive in a manner similar to the term “comprising.”

As used herein, the terms “treatment”, “treating”, and the like, referto administering an agent, or carrying out a procedure, for the purposesof obtaining an effect. In some embodiments, the effect is prophylacticin terms of completely or partially preventing a disease or symptomthereof. In some embodiments, the effect is therapeutic in terms ofaffecting a partial or complete cure for a disease or symptoms of thedisease.

As used herein, the term “antibody” refers to intact antibodies;antibody fragments, provided that they exhibit the desired biologicalactivity (e.g. epitope binding); monoclonal antibodies; polyclonalantibodies; monospecific antibodies; multi-specific antibodies (e.g.,bispecific antibodies); and antibody-like proteins.

As used herein, the term “antibody variable domain” refers to theportions of the light and heavy chains of an antibody that include aminoacid sequences of complementary determining regions (CDRs, e.g., CDR L1,CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3) and framework regions (FRs).

As used herein, the term “linker” refers to a linkage between twoelements, e.g., protein domains. In some embodiments, a linker can be acovalent bond or a spacer. The term “spacer” refers to a moiety (e.g., apolyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a1-200 amino acid sequence) occurring between two polypeptides orpolypeptide domains to provide space or flexibility (or both space andflexibility) between the two polypeptides or polypeptide domains. Insome embodiments, an amino acid spacer is part of the primary sequenceof a polypeptide (e.g., joined to the spaced polypeptides or polypeptidedomains via the polypeptide backbone).

As used herein, the term “effective amount” refers to an amount of apolypeptide or a pharmaceutical composition containing a polypeptidedescribed herein, e.g., a polypeptide having a SIRPα D1 domain orvariant thereof, that is sufficient and effective in achieving a desiredtherapeutic effect in treating a patient having a disease, such as acancer, e.g., solid tumor or hematological cancer. In some embodiments,an effective amount of polypeptide will avoid adverse side effects.

As used herein, the term “pharmaceutical composition” refers to amedicinal or pharmaceutical formulation that includes an activeingredient as well as excipients or diluents (or both excipients anddiluents) and enables the active ingredient to be administered bysuitable methods of administration. In some embodiments, thepharmaceutical compositions disclosed herein include pharmaceuticallyacceptable components that are compatible with the polypeptide. In someembodiments, the pharmaceutical composition is in tablet or capsule formfor oral administration or in aqueous form for intravenous orsubcutaneous administration, for example by injection.

As used herein, the terms “subject,” “individual,” and “patient” areused interchangeably to refer to a vertebrate, for example, a mammal.Mammals include, but are not limited to, murines, simians, humans, farmanimals, sport animals, and pets. Tissues, cells, and their progeny of abiological entity obtained in vivo or cultured in vitro are alsoencompassed. None of the terms entail supervision of a medicalprofessional.

As used herein, the term “affinity” or “binding affinity” refers to thestrength of the binding interaction between two molecules. Generally,binding affinity refers to the strength of the sum total of non-covalentinteractions between a molecule and its binding partner, such as a SIRPαD1 domain variant and CD47. Unless indicated otherwise, binding affinityrefers to intrinsic binding affinity, which reflects a 1:1 interactionbetween members of a binding pair. The binding affinity between twomolecules is commonly described by the dissociation constant (KD) or theassociation constant (KA). Two molecules that have low binding affinityfor each other generally bind slowly, tend to dissociate easily, andexhibit a large KD. Two molecules that have high affinity for each othergenerally bind readily, tend to remain bound longer, and exhibit a smallKD. In some embodiments, the KD of two interacting molecules isdetermined using known methods and techniques, e.g., surface plasmonresonance (SPR). KD can be calculated as the ratio of koff/kon.

As used herein, the term “K_(D) less than” refers to a numericallysmaller K_(D) value and an increasing binding affinity relative to therecited KD value. As used herein, the term “KD greater than” refers to anumerically larger KD value and a decreasing binding affinity relativeto the recited KD value.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual.

Overview

Provided herein are methods of treating cancer in an individual (e.g., ahuman individual) that comprise administering to the individual (a) apolypeptide (e.g., a fusion polypeptide) that comprises a SIRPα D1domain variant and an Fc domain variant and (b) a therapeutic antibody.In some embodiments, the polypeptide comprises any one of the SIRPα D1domain variants described herein (unless otherwise specified).

In some embodiments, provided is a method of treating non-small celllung cancer (NSCLC) in an individual, comprising administering to theindividual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-PD-1antibody, wherein the SIRPα D1 domain variant comprises the amino acidsequence of SEQ ID NO: 81 or SEQ ID NO: 85, wherein the Fc domainvariant is (i) a human IgG Fc region comprising L234A, L235A, G237A, andN297A mutations, wherein numbering is according to the EU index ofKabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297Amutations, wherein numbering is according to the EU index of Kabat;(iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, anddelG236 mutations, wherein numbering is according to the EU index ofKabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, delG236, and N297A mutations, wherein numbering is according tothe EU index of Kabat; and wherein the individual is a human. In someembodiments, the NSCLC in the individual has progressed on a priorimmune checkpoint inhibitor (CPI) therapy and/or has a PD-L1 tumorproportion score (TPS) of less than 50%. In some embodiments, theindividual has not received prior CPI therapy. In some embodiments, theindividual is PD-L1 negative. In some embodiments, the individual isPD-L1 positive.

In some embodiments, provided is a method of treating head and necksquamous cell carcinoma (HNSCC) in an individual, comprisingadministering to the individual an effective amount of (a) a polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant, and (b)an anti-PD-1 antibody, wherein the SIRPα D1 domain variant comprises theamino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fcdomain variant is (i) a human IgG1 Fc region comprising L234A, L235A,G237A, and N297A mutations, wherein numbering is according to the EUindex of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, andN297A mutations, wherein numbering is according to the EU index ofKabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, and delG236 mutations, wherein numbering is according to the EUindex of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, deG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; and wherein the HNSCC in theindividual has progressed while on a prior platinum therapy or after theplatinum therapy, and wherein the individual is a human. In someembodiments, the individual received prior immune checkpoint inhibitor(CPI) therapy (e.g., treatment with an immune checkpoint inhibitordescribed herein). In some embodiments, the individual has not receivedprior CPI therapy. In some embodiments, the individual is PD-L1negative. In some embodiments, the individual is PD-L1 positive.

In some embodiments an individual is “PD-L1 negative” if the individualhas a cancer that does not express PD-L1 biomarker or expresses very lowlevels of PD-L1. In some embodiments, the individual is “PD-L1 negative”or has a “PD-L1 negative cancer” if PD-L1 expression (e.g., proteinexpression) is not detected on (or in) tumor cells (TC) in a sample fromthe individual, if PD-L1 expression (e.g., protein expression) is notdetected on (or in) tumor-infiltrating immune cells (IC) in a samplefrom the individual, or if PD-L1 expression (e.g., protein expression)is detected at very low levels on (or in) TC and/or IC in a sample fromthe individual. In some embodiments, the individual is PD-L1 negativeif, e.g., 0%, less than about 1%, less than about 5%, or less than about10% of the tumor cells (TC) and/or tumor-infiltrating immune cells (IC)in a sample obtained from the individual express PD-L1, as determinedusing an assay (e.g., an assay described herein) for determining thePD-L1 status of an individual. In some embodiments, an individual ortumor may be considered PD-L1 negative because it has no T-cellinfiltrates. Such assays are known and routinely used by medicalprofessionals.

In some embodiments, an individual is “PD-L1 positive” if the individualhas a cancer that expresses (has been shown to express e.g., in adiagnostic test) PD-L1 biomarker. In some embodiments, such individualis “PD-L1 positive” or has cancer that is a “PD-L1 positive cancer.” Insome embodiments, the individual is “PD-L1 positive” or has a “PD-L1positive cancer” if PD-L1 expression (e.g., protein expression) isdetected on (or in) tumor cells (TC) in a sample from the individual, orif PD-L1 expression (e.g., protein expression) is detected on (or in)tumor-infiltrating immune cells (IC) in a sample from the individual. Insome embodiments, the individual's TC and/or IC express low levels ofPD-L1 biomarker. In some embodiments, the individual's TC and/or ICexpress high levels PD-L1 biomarker. In some embodiments, the individualis “PD-L1 positive” or has cancer that is a “PD-L1 positive cancer” ifthe PD-L1 biomarker is present (e.g., detected) in more than 0% of asample, in at least 1% of a sample, in at least 5% of a sample, or in atleast 10% of a sample from the individual (e.g., a sample from theindividual that contains the individual's TC and/or IC), as determinedusing an assay (e.g., an assay described herein) for determining thePD-L1 status of an individual. Such assays are known and routinely usedby medical professionals. In some embodiments, an individual is “PD-L”positive or has a “PD-L1 positive cancer” if the individual's tumorproportion score (TPS) is ≥50% (i.e., if ≥50% of the viable tumor cellsin a sample from the individual express PD-L1, e.g., at any level).

In some embodiments, provided is a method of treating HER2-positivegastric/gastroesophageal junction (GEJ) cancer in an individual,comprising administering to the individual an effective amount of (a) apolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant, and (b) an anti-HER2 antibody, wherein the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the gastric/GEJ cancer in the individual has progressedfollowing a prior treatment with a fluoropyrimidine-based therapy and/ora prior treatment with an anti-HER2 antibody, and wherein the individualis a human.

In some embodiments, provided is a method of treating aggressivenon-Hodgkin lymphoma or “NHL” (e.g., de novo or transformed diffuselarge B cell lymphoma (DLBCL) or mantle cell lymphoma) in an individual,comprising administering to the individual an effective amount of (a) apolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant, and (b) an anti-CD20 antibody, wherein the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and deG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the aggressive NHL in the individual is relapsed and/orrefractory to a prior treatment for aggressive NHL and there is noavailable curative therapy, and wherein the individual is a human. Insome embodiments, the relapsed/refractory aggressive NHL isrelapsed/refractory DLBCL (e.g., de novo or transformed DLBCL). In someembodiments, the a relapsed/refractory aggressive NHL isrelapsed/refractory mantle cell lymphoma (MCL).

In some embodiments, provided is a method of treating indolent lymphomain an individual, comprising administering to the individual aneffective amount of (a) a polypeptide comprising a SIRPα D1 domainvariant and an Fc domain variant, and (b) an anti-CD20 antibody, whereinthe SIRPα D1 domain variant comprises the amino acid sequence of SEQ IDNO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a humanIgG Fc region comprising L234A, L235A, G237A, and N297A mutations,wherein numbering is according to the EU index of Kabat; (ii) a humanIgG2 Fc region comprising A330S, P331S, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (iii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations,wherein numbering is according to the EU index of Kabat; or (iv) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the indolent lymphoma in the individual is relapsed and/orrefractory to a prior treatment for indolent lymphoma, and wherein theindividual is a human. In some embodiments, the indolent lymphoma (suchas a relapsed/refractory indolent lymphoma) is a non-Hodgkin lymphoma(NHL), e.g., a relapsed/refractory indolent NHL. In some embodiments,the indolent NHL (e.g., relapsed/refractory NHL) is a follicularlymphoma (e.g., a relapsed/refractory follicular lymphoma). In someembodiments, the indolent NHL (e.g., relapsed/refractory NHL) is amarginal zone lymphoma (e.g., a relapsed/refractory marginal zonelymphoma).

Further details regarding the methods of treatment and polypeptidescomprising a SIRP D1 domain variant and an Fc domain variant aredescribed below. See also U.S. Pat. No. 10,259,859, the contents ofwhich are incorporated by reference herein in their entirety.

Signal-Regulatory Protein α (SIRP-α) D1 Domain and Variants Thereof

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRPα D1domain, or a fragment thereof, that comprises an amino acid mutation atresidue 80 relative to a wild-type SIRPα D1 domain; and at least oneadditional amino acid mutation relative to a wild-type SIRPα D1 domainat a residue selected from the group consisting of residue 6, residue27, residue 31, residue 47, residue 53, residue 54, residue 56, residue66, and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc domain variants, wherein an Fc domain variant dimer comprises twoFc domain variants, wherein each Fc domain variant independently isselected from (i) a human IgG1 Fc region consisting of mutations L234A,L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting ofmutations A330S, P331S and N297A; or (iii) a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A.

Signal-regulatory protein α (“SIRP-α” or “SIRP-alpha”) is atransmembrane glycoprotein belonging to the Ig superfamily that iswidely expressed on the membrane of myeloid cells. SIRPα interacts withCD47, a protein broadly expressed on many cell types in the body. Theinteraction of SIRPα with CD47 prevents engulfment of “self” cells,which can otherwise be recognized by the immune system. It has beenobserved that high CD47 expression on tumor cells can act, in acutemyeloid leukemia and several solid tumor cancers, as a negativeprognostic factor for survival.

Native SIRPα comprises 3 highly homologous immunoglobulin (Ig)-likeextracellular domains—D1, D2, and D3. The SIRPα D1 domain (“D1 domain”)refers to the membrane distal, extracellular domain of SIRPα andmediates binding of SIRPα to CD47. As used herein, the term “SIRPαpolypeptide” refers to any SIRPα polypeptide or fragment thereof that iscapable of binding to CD47. There are at least ten variants of wild-typehuman SIRPα. Table 1 shows the amino acid sequences of the D1 domains ofthe ten naturally occurring wild-type human SIRPα D1 domain variants(SEQ ID NOs: 1-10). In some embodiments, a SIRPα polypeptide comprises aSIRPα D1 domain. In some embodiments, a SIRPα polypeptide comprises awild-type D1 domain, such as those provided in SEQ ID NOs: 1-10. In someembodiments, a SIRPα polypeptide includes a D2 or D3 domain (or both aD2 and a D3 domain) (see Table 3) of a wild-type human SIRPα.

TABLE 1 Sequences of Wild-Type SIRPα D1 Domains SEQ ID NO: DescriptionAmino Acid Sequence 1 Wild-type D1 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQdomain variant 1 WFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAG TELSVRAKPS 2 Wild-type D1EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQW domain variant 2FRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELS VRAKPS 3 Wild-type D1EEELQVIQPDKSVSVAAGESAILLCTVTSLIPVGPIQW domain variant 3FRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELS VRAKPS 4 Wild-type D1EEGLQVIQPDKSVSVAAGESAILHCTATSLIPVGPIQW domain variant 4FRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTE LSVRAKPS 5 Wild-type D1EEELQVIQPDKFVLVAAGETATLRCTATSLIPVGPIQ domain variant 5WFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAG TELSVRAKPS 6 Wild-type D1EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQ domain variant 6WFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFPIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAG TELSVRAKPS 7 Wild-type D1EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQW domain variant 7FRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELS VRGKPS 8 Wild-type D1EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQ domain variant 8WFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTEL SVRAKPS 9 domain variant 9EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQ Wild-type D1WFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDDVEFKSGAG TELSVRAKPS 10 Wild-type D1EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQW domain variant 10FRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELS VRAKPS 11 Wild-type pan-D1EEX₁LQVIQPDKX₂VX₃VAAGEX₄AX₅LX₆CTX₇TSLIP domainVGPIQWFRGAGPX₈RELIYNQKEGHFPRVTTVSX₉X₁₀TKRX₁₁NMDFX₁₂IX₁₃IX₁₄NITPADAGTYYCVKFRKGS X₁₅X₁₆DX₁₇EFKSGAGTELSVRX₁₈KPSAmino acidX₁ is E or G; X₂ is S or F; X₃ is L or S; X₄ is T or S; X₅ is Tsubstitutionsor I; X₆ is R, H, or L; X₇ is A or V; X₈ is G or A; X₉ is D orrelative toE; X₁₀ is L or S; X₁₁ is N or E or D; X₁₂ is S or P; X₁₃ is RSEQ ID NO: 11or S; X₁₄ is G or S; X₁₅ is P or absent; X₁₆ is D or P; X₁₇ isV or T; and X₁₈ is A or G

As used herein, the term “SIRPα D1 domain variant” refers to apolypeptide comprising a SIRPα D1 domain or a CD47-binding portion of aSIRPα polypeptide that has a higher affinity to CD47 than wild-typeSIRPα. A SIRPα D1 domain variant comprises at least one amino acidsubstitution, deletion, or insertion (or a combination thereof) relativeto a wild-type SIRPα.

In some embodiments, SIRPα D1 domain variants disclosed herein comprisea SIRPα D1 domain or variant thereof. In some embodiments, a SIRPα D1domain variant comprises one or more amino acid substitutions,insertions, additions, or deletions relative to a wild-type D1 domainshown in SEQ ID NOs: 1-10. Table 2 lists exemplary amino acidsubstitutions in each SIRPα D1 domain variant (SEQ ID NOs: 13-22). Insome embodiments, the SIRPα D1 domain polypeptide or SIRPα D1 domainvariant comprises a fragment of the D1 domain. In some embodiments, theSIRPα polypeptide fragment or SIRPα D1 domain variant fragment comprisesan amino acid sequence of less than 10 amino acids in length, about 10amino acids in length, about 20 amino acids in length, about 30 aminoacids in length, about 40 amino acids in length, about 50 amino acids inlength, about 60 amino acids in length, about 70 amino acids in length,about 80 amino acids in length, about 90 amino acids in length, about100 amino acids in length, or more than about 100 amino acids in length.In some embodiments, the SIRPα D1 domain fragments retain the ability tobind to CD47.

In some embodiments, a polypeptide of the disclosure comprising a SIRPαD1 domain variant binds with higher binding affinity to CD47 than awild-type human SIRPα D1 domain. In some embodiments, the SIRPα D1domain variant binds to human CD47 with at least 1-fold (e.g., at least1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold or greaterthan 5-fold) affinity than the affinity of a naturally occurring D1domain. In some embodiments, the SIRPα D1 domain variant binds to humanCD47 with at least 1-fold (e.g., at least 10-fold, 100-fold, 1000-foldor greater than 1000-fold) affinity than the affinity of a naturallyoccurring D1 domain.

As used herein, the term “optimized affinity” or “optimized bindingaffinity” refers to an optimized strength of the binding interactionbetween a polypeptide disclosed herein, including a SIRPα D1 domainvariant, and CD47. For example, in some embodiments, the polypeptidebinds primarily or with higher affinity to CD47 on cancer cells and doesnot substantially bind or binds with lower affinity to CD47 onnon-cancer cells. In some embodiments, the binding affinity between thepolypeptide and CD47 is optimized such that the interaction does notcause clinically relevant toxicity or decreases toxicity compared to avariant which binds with maximal affinity. In some embodiments, in orderto achieve an optimized binding affinity between a polypeptide providedherein and CD47, the polypeptide including a SIRPα D1 domain variant isdeveloped to have a lower binding affinity to CD47 than which ismaximally achievable. In some embodiments, the SIRPα D domain variantsdisclosed herein cross react with rodent, non-human primate (NHP), andhuman CD47.

As used herein, the term “immunogenicity” refers to the property of aprotein (e.g., a therapeutic protein) which causes an immune response inthe host as though it is a foreign antigen. The immunogenicity of aprotein can be assayed in vitro in a variety of different ways, such asthrough in vitro T-cell proliferation assays.

As used herein, the term “minimal immunogenicity” refers to animmunogenicity of a protein (e.g., a therapeutic protein) that has beenmodified, e.g., through amino acid substitutions, to be lower (e.g., atleast 10%, 25%, 50%, or 100% lower) than the immunogenicity before theamino acid substitutions are introduced (e.g., an unmodified protein).In some embodiments, a protein (e.g., a therapeutic protein) is modifiedto have minimal immunogenicity and causes no or very little host immuneresponse even though it is a foreign antigen.

In some embodiments, the SIRPα D1 domain variant demonstrates minimalimmunogenicity. In some embodiments, a SIRPα polypeptide of thedisclosure administered to a subject has the same amino acid sequence asthat of the SIRPα polypeptide in a biological sample of the subject,except for amino acid changes which increase affinity of the SIRPα D1domain variant. In some embodiments, the polypeptide variants disclosedherein lower the risk of side effects compared to anti-CD47 antibodiesor wild-type SIRPα. In some embodiments, the polypeptide variantsdisclosed herein lower the risk of anemia compared to anti-CD47antibodies or wild-type SIRPα. In some embodiments, the polypeptidevariants disclosed herein do not cause acute anemia in rodent ornon-human primates (NHP) studies.

Table 2 lists specific amino acid substitutions in a SIRPα D1 domainvariant relative to each D1 domain sequence. In some embodiments, aSIRPα D1 domain variant includes one or more (e.g., two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor more) of the substitutions listed in Table 2. In some embodiments, aSIRPα D1 domain variant includes at most fourteen amino acidsubstitutions relative to a wild-type D1 domain. In some embodiments, aSIRPα D1 domain variant includes at most ten amino acid substitutionsrelative to a wild-type D1 domain. In some embodiments, a SIRPα D1domain variant includes at most seven amino acid substitutions relativeto a wild-type D1 domain. In some embodiments, a SIRPα D1 domain variantof the disclosure has at least 90% (e.g., at least 92%, 95%, 97% orgreater than 97%) amino acid sequence identity to a sequence of awild-type D1 domain.

In some embodiments, a SIRPα D1 domain variant is a chimeric SIRPα D1domain variant that includes a portion of two or more wild-type D1domains or variants thereof (e.g., a portion of one wild-type D1 domainor variant thereof and a portion of another wild-type D1 domain orvariant thereof). In some embodiments, a chimeric SIRPα D1 domainvariant includes at least two portions (e.g., three, four, five or moreportions) of wild-type D1 domains or variants thereof, wherein each ofthe portions is from a different wild-type D1 domain. In someembodiments, a chimeric SIRPα D1 domain variant further includes one ormore amino acid substitutions listed in Table 2.

TABLE 2 Amino Acid Substitutions in a SIRPα D1 domain variant SEQ ID NO:Description Amino Acid Sequence 13 D1 domain v1EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVE X₁₄KSGAGTELSVRAKPS — Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutionsX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;relative X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, Vto SEQ ID NO: 13 14 D1 domain v2EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄K SGAGTELSVRAKPS — Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = V, I, L; X₅ = I, T, S, F;substitutionsX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = S, T, G;relative X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, Vto SEQ ID NO: 14 15 D1 domain v3EEEX₁QX₂IQPDKSVSVAAGESX₃ILLCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄K SGAGTELSVRAKPS — Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = V, I, L; X₅ = I, T, S, F;substitutionsX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = S, T, G;relative X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, Vto SEQ ID NO: 15 16 D1 domain v4EEGX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVE X₁₄KSGAGTELSVRAKPS — Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutionsX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;relative X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, Vto SEQ ID NO: 16 17 D1 domain v5EEEX₁QX₂IQPDKFVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVE X₁₄KSGAGTELSVRAKPS — Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutionsX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;relative X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, Vto SEQ ID NO: 17 18 D1 domain v6EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFPIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVE X₁₄KSGAGTELSVRAKPS — Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutionsX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;relative X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, Vto SEQ ID NO: 18 19 D1 domain v7EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄K SGAGTELSVRGKPS — Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = V, I, L; X₅ = I, T, S, F;substitutionsX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = S, T, G;relative X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, Vto SEQ ID NO: 19 20 D1 domain v8EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄ KSGAGTELSVRAKPS — Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutionsX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = S, T, G;relative X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, Vto SEQ ID NO: 20 21 D1 domain v9EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRISNITPADAGTYYCX₁₂KX₁₃RKGSPDDVE X₁₄KSGAGTELSVRAKPS — Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutionsX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;relative X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, Vto SEQ ID NO: 21 22 D1 domain v10EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄K SGAGTELSVRAKPS — Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = V, I, L; X₅ = I, T, S, F;substitutionsX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = S, T, G;relative X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, Vto SEQ ID NO: 22 23 Pan D1 domainEEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃NITPADAGTYYCX₂₄KX₂₅RKGSPDX₂₆X₂₇EX₂₈KSGAGTELSVRX₂₉KPS — Amino acidX₁ = E, G; X₂ = L, I, V; X₃ = V, L, I; X₄ = S, F; X₅ = L, S; X₆ = S,substitutionsT; X₇ = A, V; X₈ = I, T; X₉ = H, R; X₁₀ = A, V, I, L; X₁₁ = I, T, S,relative F; X₁₂ A, G; X₁₃ = E, V, L; X₁₄ = K, R; X₁₅ = E, Q; X₁₆ = H, P,to SEQ ID NO: 23R; X₁₇ = D, E; X₁₈ = S, L, T, G; X₁₉ = K, R; X₂₀ = E, D; X₂₁ = S, P;X₂₂ = S, R; X₂₃ = S, G; X₂₄ = V, I; X₂₅ = F, L, V; X₂₆ = D or absent;X₂₇ = T, V; X₂₈ = F, V; and X₂₉ = A, G

In some embodiments, a polypeptide comprises a SIRPα D1 domain variantthat comprises a sequence of:

EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVEX₁₄KSGAGTELSVRAKPS (SEQ ID NO: 13), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ is Kor R; X₁₂ is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and whereinthe variant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain that comprises the sequence of SEQ ID NO: 1.

In some embodiments, a polypeptide comprises a SIRPα D1 domain variantthat comprises a sequence of:

EEGX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVEX₁₄KSGAGTELSV RAKPS(SEQ ID NO: 16), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃ is A orV; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇ is K orR; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ is K or R; X₁₂is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and wherein the variantcomprises at least one amino acid substitution relative to a wild-typeSIRPα D1 domain that comprises the sequence of SEQ ID NO: 4.

In some embodiments, a polypeptide comprises a SIRPα D1 domain variantthat comprises a sequence of:

EEEX₁QX₂IQPDKFVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVEX₁₄KSGAGTELSVRAKPS (SEQ ID NO: 17), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ is Kor R; X₁₂ is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and whereinthe variant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain that comprises the sequence of SEQ ID NO: 5.

In some embodiments, a polypeptide comprises a SIRPα D1 domain variantthat comprises a sequence of:

EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFPIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVEX₁₄KSGAGTELSVRAKPS (SEQ ID NO: 18), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ is Kor R; X₁₂ is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and whereinthe variant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain that comprises the sequence of SEQ ID NO: 6.

In some embodiments, a polypeptide comprises a SIRPα D1 domain variantthat comprises a sequence of:

EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRISNITPADAGTYYCX₁₂KX₁₃RKGSPDDVEX₁₄KSGAGTELSVRAKPS (SEQ ID NO: 21), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ is Kor R; X₁₂ is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and whereinthe variant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain that comprises the sequence of SEQ ID NO: 9.

In any of the aforementioned embodiments, a polypeptide comprises aSIRPα D1 domain variant that comprises the sequence of any one of SEQ IDNOs: 13, 16-18, and 21, wherein X₁ is L, I, or V. In any of theaforementioned embodiments, X₂ is V, L, or, I. In any of theaforementioned embodiments, X₃ is A or V. In any of the aforementionedembodiments, X₄ is A, I, or L. In any of the aforementioned embodiments,X₅ is I, T, S, or F. In any of the aforementioned embodiments, X₆ is E,V, or L. In any of the aforementioned embodiments, X₇ is K or R. In anyof the aforementioned embodiments, X₈ is E or Q. In any of theaforementioned embodiments, X₉ is H, P, or R. In any of theaforementioned embodiments, X₁₀ is L, T, or G. In any of theaforementioned embodiments, X₁₁ is K or R. In any of the aforementionedembodiments, X₁₂ is V or I. In any of the aforementioned embodiments,X₁₃ is F, L, V. In any of the aforementioned embodiments, X₁₄ is F or V.In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than six amino acid substitutions relative to thewild-type SIRPα D1 domain that comprises the sequence of any one of SEQID NOs: 1, 4-6, and 9.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain thatcomprises the sequence of any one of SEQ ID NOs: 1, 4-6, and 9. In someembodiments, the polypeptide binds CD47 with at least 100-fold greaterbinding affinity than the wild-type SIRPα D1 domain that comprises thesequence of any one of SEQ ID NOs: 1, 4-6, and 9. In some embodiments,the polypeptide binds CD47 with at least 1000-fold greater bindingaffinity than the wild-type SIRPα D1 domain that comprises the sequenceof any one of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, a SIRPα D1domain variant polypeptide or fragment thereof binds to CD47 with aK_(D) less than 1×10⁻⁸ M, less than 5×10⁻⁹ M, less than 1×10⁻⁹ M, less5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. In someembodiments, a SIRPα D1 domain variant polypeptide or fragment thereofbinds to CD47 with a KD between about 500 nM and 100 nM, between about100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM,between about 500 pM and 100 pM, between about 100 pM and 50 pM, orbetween about 50 pM and 10 pM.

In some embodiments, a polypeptide includes a SIRPα D1 domain variantthat comprises a sequence of:

EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄KSGAGTELSVRA KPS(SEQ ID NO: 14), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃ is A orV; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇ is K orR; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is K or R; X₁₂is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and wherein the variantcomprises at least one amino acid substitution relative to a wild-typeSIRPα D1 domain that comprises the sequence of SEQ ID NO: 2.

In some embodiments, a polypeptide includes a SIRPα D1 domain variantthat comprises a sequence of:

EEEX₁QX₂IQPDKSVSVAAGESX₃ILLCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄KSGAGTELSVRA KPS(SEQ ID NO: 15), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃ is A orV; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇ is K orR; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is K or R; X₁₂is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and wherein the variantcomprises at least one amino acid substitution relative to a wild-typeSIRPα D1 domain that comprises the sequence of SEQ ID NO: 3.

In some embodiments, a polypeptide includes a SIRPα D1 domain variantthat comprises a sequence of:

EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄KSGAGTELSVRG KPS(SEQ ID NO: 19), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃ is A orV; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇ is K orR; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is K or R; X₁₂is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and wherein the variantcomprises at least one amino acid substitution relative to a wild-typeSIRPα D1 domain that comprises the sequence of SEQ ID NO: 7.

In some embodiments, a polypeptide includes a SIRPα D1 domain variantthat comprises a sequence of:

EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄KSGAGTELSVRA KPS(SEQ ID NO: 22), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃ is A orV; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇ is K orR; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is K or R; X₁₂is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and wherein the variantcomprises at least one amino acid substitution relative to a wild-typeSIRPα D1 domain that comprises the sequence of SEQ ID NO: 10.

In any of the aforementioned embodiments in this aspect of thedisclosure, the polypeptide comprises the sequence of any one of SEQ IDNOs: 14, 15, 19, and 22, wherein X₁ is L, I, or V. In any of theaforementioned embodiments, X₂ is V, L, or, I. In any of theaforementioned embodiments, X₃ is A or V. In any of the aforementionedembodiments, X₄ is V, I, or L. In any of the aforementioned embodiments,X₅ is I, T, S, or F. In any of the aforementioned embodiments, X₆ is E,V, or L. In any of the aforementioned embodiments, X₇ is K or R. In anyof the aforementioned embodiments, X₈ is E or Q. In any of theaforementioned embodiments, X₉ is H, P, or R. In any of theaforementioned embodiments, X₁₀ is S, T, or G. In any of theaforementioned embodiments, X₁₁ is K or R. In any of the aforementionedembodiments, X₁₂ is V or I. In any of the aforementioned embodiments,X₁₃ is F, L, or V. In any of the aforementioned embodiments, X₁₄ is F orV. In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than six amino acid substitutions relative to thewild-type SIRPα D1 domain that comprises the sequence of any one of SEQID NOs: 2, 3, 7, and 10.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments,the polypeptide binds CD47 with at least 100-fold greater bindingaffinity than the wild-type SIRPα D1 domain having the sequence of anyone of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptidebinds CD47 with at least 1000-fold greater binding affinity than thewild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs:2, 3, 7, and 10. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) less than1×10⁻⁸M, less than 5×10⁻⁹M, less than 1×10⁻⁹M, less 5×10⁻¹⁰ M, less than1×10⁻¹⁰ M or less than 1×10⁻¹¹M. In some embodiments, a SIRPα D1 domainvariant polypeptide or fragment thereof binds to CD47 with a K_(D)between about 500 nM and 100 nM, between about 100 nM and 50 nM, betweenabout 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nMand 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄KSGAGTELSVR AKPS(SEQ ID NO: 20), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃ is A orV; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇ is K orR; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is K or R; X₁₂is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and wherein the variantcomprises at least one amino acid substitution relative to a wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 8.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 20, wherein X₁ is L, I, or V. In any of the aforementionedembodiments in this aspect of the disclosure, X₂ is V, L, or, I. In anyof the aforementioned embodiments, X₃ is A or V. In any of theaforementioned embodiments, X₄ is A, I, or L. In any of theaforementioned embodiments, X₅ is I, T, S, or F. In any of theaforementioned embodiments, X₆ is E, V, or L. In any of theaforementioned embodiments, X₇ is K or R. In any of the aforementionedembodiments, X₈ is E or Q. In any of the aforementioned embodiments, X₉is H, P, or R. In any of the aforementioned embodiments, X₁₀ is S, T, orG. In any of the aforementioned embodiments, X₁₁ is K or R. In any ofthe aforementioned embodiments, X₁₂ is V or I. In any of theaforementioned embodiments, X₁₃ is F, L, or V. In any of theaforementioned embodiments, X₁₄ is F or V. In some embodiments, thepolypeptide of this aspect of the disclosure includes no more than sixamino acid substitutions relative to the wild-type SIRPα D1 domainhaving the sequence of SEQ ID NO: 8.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 8. In some embodiments, the polypeptide bindsCD47 with at least 100-fold greater binding affinity than the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 8. In someembodiments, the polypeptide binds CD47 with at least 1000-fold greaterbinding affinity than the wild-type SIRPα D1 domain having the sequenceof SEQ ID NO: 8. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) less than1×10⁻⁸M, less than 5×10⁻⁹ M, less than 1×10⁻⁹M, less 5×10⁻¹⁰ M, lessthan 1×10¹⁰M or less than 1×10⁻¹¹ M. In some embodiments, a SIRPα D1domain variant polypeptide or fragment thereof binds to CD47 with aK_(D) between about 500 nM and 100 nM, between about 100 nM and 50 nM,between about 50 nM and 10 nM, between about 10 nM and 5 nM, betweenabout 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pMand 100 pM, between about 100 pM and 50 pM, or between about 50 pM and10 pM.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃NITPADAGTYYCX₂₄KX₂₅RKGSPDX₂₆X₂₇EX₂₈KSGAGTELSVRX₂₉KPS(SEQ ID NO: 23), wherein X₁ is E or G; X₂ is L, I, or V; X₃ is V, L, or,I; X₄ is S or F; X₅ is L or S; X₆ is S or T; X₇ is A or V; X₈ is I or T;X₉ is H or R; X₁₀ is A, V, I, or L; X₁₁ is I, T, S, or F; X₁₂ is A or G;X₁₃ is E, V, or L; X₁₄ is K or R; X₁₅ is E or Q; X₁₆ is H, P, or R; X₁₇is D or E; X₁₈ is S, L, T, or G; X₁₉ is K or R; X₂₀ is E or D; X₂₁ is Sor P; X₂₂ is S or R; X₂₃ is S or G; X₂₄ is V or I; X₂₅ is F, L, V; X₂₆is D or absent; X₂₇ is T or V; X₂₈ is F or V; and X₂₉ is A or G; andwherein the variant comprises at least one amino acid substitutionrelative to a wild-type SIRPα D1 domain having the sequence of any oneof SEQ ID NOs: 1-10.

In any of the aforementioned embodiments in this aspect of thedisclosure, X₂ is L, I, or V. In any of the aforementioned embodiments,X₃ is V, L, or, I. In any of the aforementioned embodiments, X₄ is S orF. In any of the aforementioned embodiments, X₅ is L or S. In any of theaforementioned embodiments, X₆ is S or T. In any of the aforementionedembodiments, X₇ is A or V. In any of the aforementioned embodiments, X₈is I or T. In any of the aforementioned embodiments, X₉ is H or R. Inany of the aforementioned embodiments, X₁₀ is A, V, I, or L. In any ofthe aforementioned embodiments, X₁₁ is I, T, S, or F. In any of theaforementioned embodiments, X₁₂ is A or G. In any of the aforementionedembodiments, X₁₃ is E, V, or L. In any of the aforementionedembodiments, X₁₄ is K or R. In any of the aforementioned embodiments, X₅is E or Q. In any of the aforementioned embodiments, X₁₆ is H, P, or R.In any of the aforementioned embodiments, X₁₇ is D or E. In any of theaforementioned embodiments, X₁₈ is S, L, T, or G. In any of theaforementioned embodiments, X₁₉ is K or R. In any of the aforementionedembodiments, X₂₀ is E or D. In any of the aforementioned embodiments,X₂₁ is S or P. In any of the aforementioned embodiments, X₂₂ is S or R.In any of the aforementioned embodiments, X₂₃ is S or G. In any of theaforementioned embodiments, X₂₄ is V or I. In any of the aforementionedembodiments, X₂₅ is F, L, V. In any of the aforementioned embodiments,X₂₆ is D or absent. In any of the aforementioned embodiments, X₂₇ is Tor V. In any of the aforementioned embodiments, X₂s is F or V. In any ofthe aforementioned embodiments, X₂₉ is A or G. In some embodiments, thepolypeptide of this aspect of the disclosure includes no more than sixamino acid substitutions relative to the wild-type SIRPα D1 domainhaving the sequence of any one of SEQ ID NOs: 1-10.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of any one of SEQ ID NOs: 1-10. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRPα D1 domain having the sequence of any one of SEQID NOs: 1-10. In some embodiments, the polypeptide binds CD47 with atleast 1000-fold greater binding affinity than the wild-type SIRPα D1domain having the sequence of any one of SEQ ID NOs: 1-10. In someembodiments, a SIRPα D1 domain variant polypeptide or fragment thereofbinds to CD47 with a K_(D) less than 1×10⁻⁸ M, less than 5×10⁻⁹ M, lessthan 1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹M. In some embodiments, a SIRPα D1 domain variant polypeptide orfragment thereof binds to CD47 with a K_(D) between about 500 nM and 100nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM,between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pMand 50 pM, or between about 50 pM and 10 pM.

In some embodiments, a polypeptide of the disclosure including a SIRPαD1 domain variant further comprises a D2 domain having the sequence ofSEQ ID NO: 24, a D3 domain having the sequence of SEQ ID NO: 25, or a D2domain having the sequence of SEQ ID NO: 24 and a D3 domain having thesequence of SEQ ID NO: 25 of a wild-type human SIRPα as shown in Table3. In some embodiments, the SIRPα D1 domain variant further comprises afragment or variant of a D2 domain or a fragment or variant of a D3domain. In some embodiments, the SIRPα D1 domain variant furthercomprises a fragment or variant of a D2 domain and a fragment or variantof a D3 domain. In some embodiments, a SIRPα D1 domain variant is joinedto a D2 or D3 domain by way of a linker. In some embodiments, a SIRPα D1domain variant is joined to a D2 and D3 domain by way of a linker.

TABLE 3 Amino Acid Sequences of SIRPα D2 and D3 Domains SEQ ID NO:Description Amino Acid Sequence 24 SIRPα D2 APVVSGPAARATPQHTVSFTCESHGFdomain SPRDITLKWFKNGNELSDFQTNVDPV GESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIR 25 SIRPα D3 VPPTLEVTQQPVRAENQVNVTCQVR domainKFYPQRLQLTWLENGNVSRTETAST VTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVS

In some embodiments, a polypeptide of the disclosure including a SIRPαD1 domain variant is attached to an Fc domain variant in order toimprove the pharmacokinetic properties of the polypeptide, e.g.,increase serum half-life. In some embodiments, a SIRPα D1 domain variantis attached to an Fc domain variant that is unable to dimerize. In someembodiments, Fc domain variants serve to increase the serum half-life ofthe polypeptides described herein. In some embodiments, a polypeptide ofthe disclosure including a SIRPα D1 domain variant does not include thesequence of any one of SEQ ID NOs: 26-36 shown in Table 4.

TABLE 4 SEQ ID NO: Amino Acid Sequence 26EEELQVIQPDKSVSVAAGESAILHCTITSLIPVGPIQWFRGAGPARELIYNQREGHFPRVTTVSETTRRENMDFSISISNITPADAGTYYCVKFRKGSPDTEVKSGAGTELSVRAKPS 27EEEVQVIQPDKSVSVAAGESAILHCTLTSLIPVGPIQWFRGAGPARVLIYNQRQGHFPRVTTVSEGTRRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKPS 28EEEVQIIQPDKSVSVAAGESVILHCTITSLTPVGPIQWFRGAGPARLLIYNQREGPFPRVTTVSETTRRENMDFSISISNITPADAGTYYCVKLRKGSPDTEFKSGAGTELSVRAKPS 29EEELQIIQPDKSVSVAAGESAILHCTITSLSPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSEGTKRENMDFSISISNITPADAGTYYCIKLRKGSPDTEFKSGAGTELSVRAKPS 30EEEIQVIQPDKSVSVAAGESVIIHCTVTSLFPVGPIQWFRGAGPARVLIYNQRQGRFPRVTTVSEGTKRENMDFSISISNITPADAGTYYCVKVRKGSPDTEVKSGAGTELSVRAKPS 31EEEVQIIQPDKSVSVAAGESIILHCTVTSLFPVGPIQWFRGAGPARVLIYNQREGRFPRVTTVSEGTRRENMDFSISISNITPADAGTYYCIKLRKGSPDTEFKSGAGTELSVRAKPS 32EEEVQLIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARVLIYNQREGPFPRVTTVSEGTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEVKSGAGTELSVRAKPS 33EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPS 34EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARLLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS 35EEEVQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQKQGPFPRVTTISETTRRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKPS 36EEELQIIQPDKSVSVAAGESAILHCTITSLTPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSEGTRRENMDFSISISNITPADAGTYYCIKFRKGSPDTEVKSGAGTELSVRAKPS

In some embodiments, the polypeptides and polypeptide constructsdescribed herein are utilized in vitro for binding assays, such asimmune assays. For example, in some embodiments, the polypeptides andpolypeptide constructs described herein are utilized in liquid phase orbound to a solid phase carrier. In some embodiments, polypeptidesutilized for immunoassays are detectably labeled in various ways.

In some embodiments, polypeptides and polypeptide constructs describedherein are bound to various carriers and used to detect the presence ofspecific antigen expressing cells. Examples of carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses, andmagnetite. The nature of the carrier can be either soluble or insoluble.

Various different labels and methods of labeling are known. Examples oflabels include enzymes, radioisotopes, fluorescent compounds, colloidalmetals, chemiluminescent compounds, and bio-luminescent compounds.Various techniques for binding labels to polypeptides disclosed hereinare available.

In some embodiments, the polypeptides are coupled to low molecularweight haptens. These haptens are then specifically detected by means ofa second reaction. For example, in some embodiments, the hapten biotinis used with avidin or the haptens dinitrophenol, pyridoxal, orfluorescein are detected with specific anti-hapten antibodies (e.g.,anti-dinitrophenol antibodies, anti-pyridoxal antibodies, andanti-fluorescein antibodies respectively).

SIRPα D1 Domain Variants with Altered Glycosylation Patterns

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRPα D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRPα D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRPα D1 domain at a residueselected from the group consisting of: residue 6, residue 27, residue31, residue 47, residue 53, residue 54, residue 56, residue 66, andresidue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc domain variant, wherein an Fc domain variant dimer comprises twoFc domain variants, wherein each Fc domain variant independently isselected from (i) a human IgG1 Fc region consisting of mutations L234A,L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting ofmutations A330S, P331S and N297A; or (iii) a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A.

In some embodiments, a polypeptide in a composition disclosed hereincomprises a SIRPα D1 domain variant that has reduced or minimalglycosylation. The D1 domain of each of the ten wild-type human SIRPαproteins (SEQ ID NOs: 1-10 in Table 1) contains a single potentialN-linked glycosylation site at amino acid N80 in the sequence N80ITP.Expression of a SIRPα D1 domain in Chinese Hamster Ovary (CHO) cellsresults in a major band of 16 kDa (non-glycosylated) and a minor band ofhigher molecular weight that was removed by Endo Hf. Endo Hf is arecombinant protein fusion of Endoglycosidase H and maltose bindingprotein. Endo Hf cleaves within the chitobiose core of high mannose andsome hybrid oligosaccharides from N-linked glycoproteins. This impliesthat a proline at amino acid position 83 can reduce the efficiency ofglycosylation, leading to a protein with different degrees ofglycosylation and therefore heterogeneity. For drug development,heterogeneity can give rise to challenges in process development.Therefore, to investigate the possibility of generating homogenous,non-glycosylated forms of SIRPα D1 domain variants, in some embodiments,amino acid N80 of a SIRPα D1 variant is mutated to Ala. In someembodiments, to make a non-glycosylated, SIRPα D1 domain variant, aminoacid N80 in a SIRPα D1 domain variant is replaced by any amino acid,including any naturally and non-naturally occurring amino acid, e.g.,N80A and N80Q. In some embodiments, a SIRPα D1 domain variant comprisesan N80A mutation and at least 1 additional mutation (e.g., at least 2,3, 4, 5, 6, 7, 8, 9, or 10 additional mutations or more). In someembodiments, the additional mutation is in the CD47 binding site. Insome embodiments, the additional mutation is in the hydrophobic core ofthe D1 domain.

In some embodiments, a polypeptide in a composition disclosed hereinincludes a SIRPα D1 domain variant that has increased glycosylationrelative to a wild-type SIRPα D1 domain. Another option to increasehomogeneity of the final product is to enhance the efficiency ofglycosylation at amino acid N80 and generate SIRPα D1 domain variantswith increased glycosylation relative to a wild-type. In someembodiments, the amino acid P83 in the sequence NITP83 affects thedegree of glycosylation at amino acid N80. In some embodiments, changingP83 to any amino acid increases the efficiency of glycosylation at N80.In some embodiments, amino acid P83 in a SIRPα D1 domain variant isreplaced by any amino acid, including naturally and non-naturally aminoacids, e.g., P83V, P83A, P83I, and P83L. In some embodiments, apolypeptide of the disclosure is expressed in a cell that is optimizednot to glycosylate proteins that are expressed by such cell, for exampleby genetic engineering of the cell line (e.g., genetically engineeredyeast or mammalian host) or modifications of cell culture conditionssuch as addition of kifunensine or by using a naturallynon-glycosylating host such as a prokaryote (E. coli, etc.).

Table 5 lists specific amino acid substitutions in a SIRPα D1 domainvariant relative to each D1 domain variant sequence. In someembodiments, a SIRPα D1 domain variant includes one or more (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or more) of the substitutions listed in Table 5. Insome embodiments, the SIRPα D1 domain variants are not glycosylated orare minimally glycosylated. In some embodiments, the SIRPα D1 domainvariants are fully glycosylated or almost fully glycosylated. In someembodiments, a SIRPα D1 domain variant includes at most fourteen aminoacid substitutions relative to a wild-type D1 domain. In someembodiments, a SIRPα D1 domain variant includes at most ten amino acidsubstitutions relative to a wild-type D1 domain. In some embodiments, aSIRPα D1 domain variant includes at most seven amino acid substitutionsrelative to a wild-type D1 domain. In some embodiments, a SIRPα D1domain variant of the disclosure has at least 90% (e.g., at least 92%,95%, 97% or greater than 97%) amino acid sequence identity to a sequenceof a wild-type D1 domain.

In some embodiments, a SIRPα D1 domain variant is a chimeric SIRPα D1domain variant that includes a portion of two or more wild-type D1domains or variants thereof (e.g., a portion of one wild-type D1 domainor variant thereof and a portion of another wild-type D1 domain orvariant thereof). In some embodiments, a chimeric SIRPα D1 domainvariant includes at least two portions (e.g., three, four, five or moreportions) of wild-type D1 domains or variants thereof, wherein each ofthe portions is from a different wild-type D1 domain. In someembodiments, a chimeric SIRPα D1 domain variant further includes one ormore amino acid substitutions listed in Table 5.

TABLE 5 Amino Acid Substitutions in a SIRPα Dl domain variant SEQ ID NO:Description Amino Acid Sequence 37 D1 domain v1EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDD VEX₁₆KSGAGTELSVRAKPS —Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutions relativeX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;to SEQ ID NO: 37X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R,S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 38 D1 domain v2EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆ KSGAGTELSVRAKPS —Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = V, I, L; X₅ = I, T, S, F;substitutions relativeX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = S, T, G;to SEQ ID NO: 38X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R,S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 39 D1 domain v3EEEX₁QX₂IQPDKSVSVAAGESX₃ILLCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆ KSGAGTELSVRAKPS —Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = V, I, L; X₅ = I, T, S, F;substitutions relativeX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = S, T, G;to SEQ ID NO: 39X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R,S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 40 D1 domain v4EEGX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDD VEX₁₆KSGAGTELSVRAKPS —Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutions relativeX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;to SEQ ID NO: 40X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R,S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 41 D1 domain v5EEEX₁QX₂IQPDKFVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDD VEX₁₆KSGAGTELSVRAKPS —Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutions relativeX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;to SEQ ID NO: 41X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R,S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 42 D1 domain v6EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFPIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDD VEX₁₆KSGAGTELSVRAKPS —Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutions relativeX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;to SEQ ID NO: 42X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R,S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 43 D1 domain v7EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆ KSGAGTELSVRGKPS —Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = V, I, L; X₅ = I, T, S, F;substitutions relativeX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = S, T, G;to SEQ ID NO: 43X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R,S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 44 D1 domain v8EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTE X₁₆KSGAGTELSVRAKPS —Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutions relativeX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = S, T, G;to SEQ ID NO: 44X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R,S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 45 D1 domain v9EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDD VEX₁₆KSGAGTELSVRAKPS —Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = A, I, L; X₅ = I, T, S, F;substitutions relativeX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;to SEQ ID NO: 45X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R,S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 46 D1 domain v10EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆ KSGAGTELSVRAKPS —Amino acidX₁ = L, I, V; X₂ = V, L, I; X₃ = A, V; X₄ = V, I, L; X₅ = I, T, S, F;substitutions relativeX₆ = E, V, L; X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = S, T, G;to SEQ ID NO: 46X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R,S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 47 Pan D1 domainEEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃X₂₄ITX₂₅ADAGTYYCX₂₆KX₂₇RKGSPDX₂₈X₂₉EX₃₀KSGAGTELSVRX₃₁KPS — Amino acidX₁ = E, G; X₂ = L, I, V; X₃ = V, L, I; X₄ = S, F; X₅ = L, S; X₆ = S,substitutions relativeT; X₇ = A, V; X₈ = I, T; X₉ = H, R, L; X₁₀ = A, V, I, L; X₁₁ = I, T,to SEQ ID NO: 47S, F; X₁₂ = A, G; X₁₃ = E, V, L; X₁₄ = K, R; X₁₅ = E, Q; X₁₆ = H, P,R; X₁₇ = D, E; X₁₈ = S, L, T, G; X₁₉ = K, R; X₂₀ = E, N; X₂₁ = S, P;X₂₂ = S, R; X₂₃ = S, G; X₂₄ = any amino acid; X₂₅ = any aminoacid; X₂₆ = V, I; X₂₇ = F, L, V; X₂₈ = D or absent; X₂₉ = T, V;X₃₀ = F, V; and X₃₁ = A, G 48 Pan D1 domainEEELQX₁IQPDKSVX₂VAAGEX₃AX₄LX₅CTX₆TSLX₇PVGPIQWFRGAGPX₈RX₉LIYNQX₁₀X₁₁GX₁₂FPRVTTVSX₁₃X₁₄TKRX₁₅NMDFSIX₁₆IX₁₇X₁₈ITPADAGTYYCX₁₉KFRKGX₂₀X₂₁X₂₂DX₂₃EFKSGAGTELSVRAKPS — Amino acidX₁ =V, I; X₂ =L, S; X₃ =T, S; X₄ =T, I; X₅ =R, H; X₆ =A, V,substitutions relativeI; X₇ = I, R, Y, K, F; X₈ = G, A; X₉ = E, V; X₁₀ = K, R; X₁₁ =to SEQ ID NO: 48E, D, Q; X₁₂ = H, P; X₁₃ = D, E; X₁₄ = S, L, T; X₁₅ = N, E; X₁₆ =R, S; X₁₇ = G, S; X₁₈ = N, A; X₁₉ = V, I; X₂₀ = S, I, M; X₂₁ = Por absent; X₂₂ = D, P; and X₂₃ = V, T 49 Pan D1 domainEEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉ITPADAGTYYCX₁₀KFRKGSPDDVEFKSG AGTELSVRAKPS — Amino acidX₁ = V, I, L; X₂ = A, I, V, L; X₃ = I, F, S, T; X₄ = E, V, L; X₅ = K,substitutions relativeR; X₆ = E, Q; X₇ = H, P, R; X₈ = L, T, S, G; X₉ = A; and X₁₀ = V,to SEQ ID NO: 49 I 50 Pan D1 domainEEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPIQWFRGAGPARX₄LIYNQX₅X₆GX₇FPRVTTVSEX₈TKRENMDFSISISX₉ITPADAGTYYCX₁₀KFRKGSPDTEFKSGAG TELSVRAKPS — Amino acidX₁ = V, I; X₂ = V, I; X₃ = I, F; X₄ = E, V; X₅ = K, R; X₆ = E, Q;substitutions relative X₇ = H, P; X₈ = S, T; X₉ = N, A; and X₁₀ = V, Ito SEQ ID NO: 50 51 Pan D1 domainEEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅EGX₆FPRVTTVSDX₇TKRNNMDFSIRIGX₈ITPADAGTYYCX₉KFRKGSPDDVEFKSGA GTELSVRAKPS — Amino acidX₁ = V, I; X₂ = A, I; X₃ = I, F; X₄ = E, V; X₅ = K, R; X₆ = H, P;substitutions relative X₇ = L, T; X₈ = N, A; and X₉ = V, Ito SEQ ID NO: 51 52 Pan D1 domainEEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRELIYNQX₄EGX₅FPRVTTVSDX₆TKRNNMDFSIRIGX₇ITPADAGTYYCVKFRKGSPDDVEFKSGAGT ELSVRAKPS — Amino acidX₁ = V, L, I; X₂ = A, I, L; X₃ = I, T, S, F; X₄ = K, R; X₅ = H, P, R;substitutions relative X₆ = L, T, G; and X₇ = N, A to SEQ ID NO: 52212   Pan D1 domain EEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPIQWFRGAGPARELIYNQX₄EGX₅FPRVTTVSEX₆TKRENMDFSISISX₇ITPADAGTYYCVKFRKGSPDTEFKSGAGTE LSVRAKPS — Amino acidX₁ = V, L, I; X₂ = V, I, L; X₃ = I, T, S, F; X₄ = K, R; X₅ = H, P, R;substitutions relative X₆ = S, T, G; and X₇ = N, A to SEQ ID NO: 212218   Pan D1 domain EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉X₁₀X₁₁X₁₂ADAGTYYCX₁₃KFRKGSPDDVE FKSGAGTELSVRAKPS — Amino acidX₁ = V, L, or I; X₂ = A, V, L, or I; X₃ = I, S, T, or F; X₄ = E, L,substitutions relativeor V; X₅ = K or R; X₆ = E or Q; X₇ = H, R or P; X₈ = S, G, L or T,to SEQ ID NO: 218X₉ = any amino acid; X₁₀ = any amino acid; X₁₁ = any aminoacid; X₁₂ = any amino acid; and X₁₃ = V or I 219   Pan D1 domainEEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉ITX₁₀ADAGTYYCX₁₁KFRKGSPDDVEFKS GAGTELSVRAKPS —   Amino acidX₁ = V, L or I; X₂ = A, V, L, or I; X₃ = I, S, T or F; X₄ = E, L, orsubstitutions relativeV; X₅ = K or R; X₆ = E or Q; X₇ = H, R or P; X₈ = S, G, L, or T;to SEQ ID NO: 219X₉ = N; X₁₀ = any amino acid other than P; and X₁₁ = V or I

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDDVEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 37), wherein X₁ is L, I, or V; X₂ is V, L, or, I;X₃ is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L;X₇ is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ isK or R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W,or Y; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, orY; X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEGX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDDVEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 40), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ is Kor R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain having the sequence of SEQ ID NO: 4.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEEX₁QX₂IQPDKFVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDDVEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 41), wherein X₁ is L, I, or V; X₂ is V, L, or, I;X₃ is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L;X₇ is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ isK or R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W,or Y; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, orY; X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain having the sequence of SEQ ID NO: 5.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFPIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDDVEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 42), and wherein X₁ is L, I, or V; X₂ is V, L, or,I; X₃ is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, orL; X₇ is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁is K or R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V,W, or Y; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W,or Y; X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and whereinthe variant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain having the sequence of SEQ ID NO: 6.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDDVEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 45), and wherein X₁ is L, I, or V; X₂ is V, L, or,I; X₃ is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, orL; X₇ is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁is K or R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V,W, or Y; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W,or Y; X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and whereinthe variant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain having the sequence of SEQ ID NO: 9.

In any of the aforementioned embodiments in this aspect of thedisclosure, a polypeptide includes a SIRPα D1 domain variant having asequence of any one of SEQ ID NOs: 37, 40-42, and 45, wherein X₁ is L,I, or V. In any of the aforementioned embodiments, X₂ is V, L, or, I. Inany of the aforementioned embodiments, X₃ is A or V. In any of theaforementioned embodiments, X₄ is A, I, or L. In any of theaforementioned embodiments, X₅ is I, T, S, or F. In any of theaforementioned embodiments, X₆ is E, V, or L. In any of theaforementioned embodiments, X₇ is K or R. In any of the aforementionedembodiments, X₈ is E or Q. In any of the aforementioned embodiments, X₉is H, P, or R. In any of the aforementioned embodiments, X₁₀ is L, T, orG. In any of the aforementioned embodiments, X₁₁ is K or R. In any ofthe aforementioned embodiments, X₁₂ is N, A, C, D, E, F, G, H, I, K, L,M, P, Q, R, S, T, V, W, or Y. In any of the aforementioned embodiments,X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. Inany of the aforementioned embodiments, X₁₄ is V or I. In any of theaforementioned embodiments, X₁₅ is F, L, V. In any of the aforementionedembodiments, X₁₆ is F or V.

In some embodiments, a polypeptide provided herein includes no more thanten amino acid substitutions relative to the wild-type SIRPα D1 domainhaving the sequence of any one of SEQ ID NOs: 1, 4-6, and 9. In someembodiments, the polypeptide provided herein includes no more than sevenamino acid substitutions relative to the wild-type SIRPα D1 domainhaving the sequence of any one of SEQ ID NOs: 1, 4-6, and 9.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of any one of SEQ ID NOs: 1, 4-6, and 9. In some embodiments,the polypeptide binds CD47 with at least 100-fold greater bindingaffinity than the wild-type SIRPα D1 domain having the sequence of anyone of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the polypeptidebinds CD47 with at least 1000-fold greater binding affinity than thewild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs:1, 4-6, and 9. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) less than1×10⁻⁸ M, less than 5×10⁻⁹ M, less than 1×10⁻⁹ M, less 5×10⁻¹ M, lessthan 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. In some embodiments, a SIRPα D1domain variant polypeptide or fragment thereof binds to CD47 with aK_(D) between about 500 nM and 100 nM, between about 100 nM and 50 nM,between about 50 nM and 10 nM, between about 10 nM and 5 nM, betweenabout 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pMand 100 pM, between about 100 pM and 50 pM, or between about 50 pM and10 pM.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 38), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is Kor R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEEX₁QX₂IQPDKSVSVAAGESX₃ILLCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 39), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is Kor R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant comprises at least one amino acid substitution relative to awild-type SIRPα D domain having the sequence of SEQ ID NO: 3.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆KSGAGTELSVRGKPS (SEQ ID NO: 43), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is Kor R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant comprises at least one amino acid substitution relative to awild-type SIRPα D domain having the sequence of SEQ ID NO: 7.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 46), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is Kor R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant comprises at least one amino acid substitution relative to awild-type SIRPα D1 domain having the sequence of SEQ ID NO: 10.

In any of the aforementioned embodiments in this aspect of thedisclosure, a polypeptide includes a SIRPα D1 domain variant having asequence of any one of SEQ ID NOs: 38, 39, 43, and 46, wherein X₁ is L,I, or V. In any of the aforementioned embodiments, X₂ is V, L, or, I. Inany of the aforementioned embodiments, X₃ is A or V. In any of theaforementioned embodiments, X₄ is V, I, or L. In any of theaforementioned embodiments, X₅ is I, T, S, or F. In any of theaforementioned embodiments, X₆ is E, V, or L. In any of theaforementioned embodiments, X₇ is K or R. In any of the aforementionedembodiments, X₈ is E or Q. In any of the aforementioned embodiments, X₉is H, P, or R. In any of the aforementioned embodiments, X₁₀ is S, T, orG. In any of the aforementioned embodiments, X₁₁ is K or R. In any ofthe aforementioned embodiments, X₁₂ is N, A, C, D, E, F, G, H, I, K, L,M, P, Q, R, S, T, V, W, or Y. In any of the aforementioned embodiments,X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. Inany of the aforementioned embodiments, X₁₄ is V or I. In any of theaforementioned embodiments, X₁₈ is F, L, or V. In any of theaforementioned embodiments, X₁₆ is F or V.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs:2, 3, 7, and 10. In some embodiments, a polypeptide includes a SIRPα D1domain variant having no more than seven amino acid substitutionsrelative to the wild-type SIRPα D1 domain having the sequence of any oneof SEQ ID NOs: 2, 3, 7, and 10.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments,the polypeptide binds CD47 with at least 100-fold greater bindingaffinity than the wild-type SIRPα D1 domain having the sequence of anyone of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptidebinds CD47 with at least 1000-fold greater binding affinity than thewild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs:2, 3, 7, and 10. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) less than1×10⁻⁸M, less than 5×10⁻⁹M, less than 1×10⁻⁹ M, less 5×10⁻¹⁰ M, lessthan 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. In some embodiments, a SIRPα D1domain variant polypeptide or fragment thereof binds to CD47 with aK_(D) between about 500 nM and 100 nM, between about 100 nM and 50 nM,between about 50 nM and 10 nM, between about 10 nM and 5 nM, betweenabout 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pMand 100 pM, between about 100 pM and 50 pM, or between about 50 pM and10 pM.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 44), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is Kor R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant comprises at least one amino acid substitution relative to awild-type SIRPα D domain having the sequence of SEQ ID NO: 8.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 44, wherein X₁ is L, I, or V. In any of the aforementionedembodiments in this aspect of the disclosure, X₂ is V, L, or, I. In anyof the aforementioned embodiments, X₃ is A or V. In any of theaforementioned embodiments, X₄ is A, I, or L. In any of theaforementioned embodiments, X₅ is I, T, S, or F. In any of theaforementioned embodiments, X₆ is E, V, or L. In any of theaforementioned embodiments, X₇ is K or R. In any of the aforementionedembodiments, X₈ is E or Q. In any of the aforementioned embodiments, X₉is H, P, or R. In any of the aforementioned embodiments, X₁₀ is S, T, orG. In any of the aforementioned embodiments, X₁₁ is K or R. In any ofthe aforementioned embodiments, X₁₂ is N, A, C, D, E, F, G, H, I, K, L,M, P, Q, R, S, T, V, W, or Y. In any of the aforementioned embodiments,X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. Inany of the aforementioned embodiments, X₁₄ is V or I. In any of theaforementioned embodiments, X₁₈ is F, L, or V. In any of theaforementioned embodiments, X₁₆ is F or V.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In someembodiments, a polypeptide includes a SIRPα D1 domain variant having nomore than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 8.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of SEQ ID NO: 8. In some embodiments, the polypeptide bindsCD47 with at least 100-fold greater binding affinity than the wild-typeSIRPα D1 domain having the sequence of SEQ ID NO: 8. In someembodiments, the polypeptide binds CD47 with at least 1000-fold greaterbinding affinity than the wild-type SIRPα D1 domain having the sequenceof SEQ ID NO: 8. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) less than1×10⁻⁸M, less than 5×10⁻⁹ M, less than 1×10⁻⁹M, less 5×10⁻¹⁰ M, lessthan 1×10¹⁰M or less than 1×10⁻¹¹ M. In some embodiments, a SIRPα D1domain variant polypeptide or fragment thereof binds to CD47 with aK_(D) between about 500 nM and 100 nM, between about 100 nM and 50 nM,between about 50 nM and 10 nM, between about 10 nM and 5 nM, betweenabout 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pMand 100 pM, between about 100 pM and 50 pM, or between about 50 pM and10 pM.

In another aspect, the disclosure features a polypeptide including aSIRPα D1 domain variant having a sequence of:

EEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀SLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃X₂₄ITX₂₅ADAGTYYCX₂₆KX₂₇RKGSPDX₂₈X₂₉EX₃₀KSGAGTELSVRX₃₁KPS (SEQ ID NO: 47), wherein X₁ is E or G; X₂is L, I, or V; X₃ is V, L, or, I; X₄ is S or F; X₅ is L or S; X₆ is S orT; X₇ is A or V; X₈ is I or T; X₉ is H, R, or L; X₁₀ is A, V, I, or L;X₁₁ is I, T, S, or F; X₁₂ is A or G; X₁₃ is E, V, or L; X₁₄ is K or R;X₁₅ is E or Q; X₁₆ is H, P, or R; X₁₇ is D or E; X₁₈ is S, L, T, or G;X₁₉ is K or R; X₂₀ is E or N; X₂₁ is S or P; X₂₂ is S or R; X₂₃ is S orG; X₂₄ is any amino acid; X₂₅ is any amino acid; X₂₆ is V or I; X₂₇ isF, L, V; X₂₈ is D or absent; X₂₉ is T or V; X₃₀ is F or V; and X₃₁ is Aor G; and wherein the variant comprises at least one amino acidsubstitution relative to a wild-type SIRPα D1 domain having the sequenceof any one of SEQ ID NOs: 1-10.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 47, wherein X₁ is E or G. In any of the aforementioned embodimentsin this aspect of the disclosure, X₂ is L, I, or V. In any of theaforementioned embodiments, X₃ is V, L, or, I. In any of theaforementioned embodiments, X₄ is S or F. In any of the aforementionedembodiments, X₅ is L or S. In any of the aforementioned embodiments, X₆is S or T. In any of the aforementioned embodiments, X₇ is A or V. Inany of the aforementioned embodiments, X₈ is I or T. In any of theaforementioned embodiments, X₉ is H or R. In any of the aforementionedembodiments, X₁₀ is A, V, I, or L. In any of the aforementionedembodiments, X₁₁ is I, T, S, or F. In any of the aforementionedembodiments, X₁₂ is A or G. In any of the aforementioned embodiments,X₁₃ is E, V, or L. In any of the aforementioned embodiments, X₁₄ is K orR. In any of the aforementioned embodiments, X₁₅ is E or Q. In any ofthe aforementioned embodiments, X₁₆ is H, P, or R. In any of theaforementioned embodiments, X₁₇ is D or E. In any of the aforementionedembodiments, X₁₈ is S, L, T, or G. In any of the aforementionedembodiments, X₁₉ is K or R. In any of the aforementioned embodiments, X₂is E or N. In any of the aforementioned embodiments, X₂₁ is S or P. Inany of the aforementioned embodiments, X₂₂ is S or R. In any of theaforementioned embodiments, X₂₃ is S or G. In any of the aforementionedembodiments, X₂₄ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T,V, W, or Y. In any of the aforementioned embodiments, X₂₅ is P, A, C, D,E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In any of theaforementioned embodiments, X₂₆ is V or I. In any of the aforementionedembodiments, X₂₇ is F, L, V. In any of the aforementioned embodiments,X₂₈ is D or absent. In any of the aforementioned embodiments, X₂₉ is Tor V. In any of the aforementioned embodiments, X₃₀ is F or V. In any ofthe aforementioned embodiments, X₃₁ is A or G.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs:1-10. In some embodiments, the polypeptide of this aspect of thedisclosure includes no more than seven amino acid substitutions relativeto the wild-type SIRPα D1 domain having the sequence of any one of SEQID NOs: 1-10.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of any one of SEQ ID NOs: 1-10. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRPα D1 domain having the sequence of any one of SEQID NOs: 1-10. In some embodiments, the polypeptide binds CD47 with atleast 1000-fold greater binding affinity than the wild-type SIRPα D1domain having the sequence of any one of SEQ ID NOs: 1-10. In someembodiments, a SIRPα D1 domain variant polypeptide or fragment thereofbinds to CD47 with a K_(D) less than 1×10⁻⁸ M, less than 5×10⁻⁹ M, lessthan 1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹M. In some embodiments, a SIRPα D1 domain variant polypeptide orfragment thereof binds to CD47 with a K_(D) between about 500 nM and 100nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM,between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pMand 50 pM, or between about 50 pM and 10 pM.

In some embodiments, a polypeptide includes a SIRPα D1 domain varianthaving a sequence of:

EEELQX₁IQPDKSVX₂VAAGEX₃AX₄LX₅CTX₆TSLX₇PVGPIQWFRGAGPX₈RX₉LIYNQX₁₀X₁₁GX₁₂FPRVTTVSX₁₃X₁₄TKRX₁₅NMDFSIX₁₆IX₁₇X₁₈ITPADAGTYYCX₁₉KFRKGX₂₀X₂₁X₂₂DX₂₃EFKSGAGTELSVRAKPS(SEQ ID NO: 48), wherein X₁ is V or I; X₂ is L or S; X₃ is T or S; X₄ isT or I; X₅ is R or H; X₆ is A, V, or I; X₇ is I, R, Y, K or F; X₈ is Gor A; X₉ is E or V; X₁₀ is K or R; X₁₁ is E, D or Q; X₁₂ is H or P; X₁₃is D or E; X₁₄ is S, L or T; X₁₅ is N or E; X₁₆ is R or S; X₁₇ is G orS; X₁₈ is N or A; X₁₉ is V or I; X₂₀ is S, I or M; X₂₁ is P or absent;X₂₂ is D or P; and X₂₃ is V or T, or a fragment thereof.

In another aspect, the disclosure features a polypeptide including aSIRPα D1 domain variant having a sequence of:

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉ITPADAGTYYCX₁₀KFRKGSPDDVEFKSGAGTELSVRAKP S (SEQID NO: 49), wherein X₁ is V, L, or I; X₂ is A, I, V, or L; X₃ is I, F,S, or T; X₄ is E, V, or L; X₅ is K or R; X₆ is E or Q; X₇ is H, P, or R;X₈ is L, T, S, or G; X₉ is A; and X₁₀ is V or I; and wherein the variantcomprises at least one amino acid substitution relative to a wild-typeSIRPα D domain having the sequence of any one of SEQ ID NO: 1.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 49, wherein X₁ is V, L or I. In any of the aforementionedembodiments in this aspect of the disclosure, X₂ is A, I, V, or L. Inany of the aforementioned embodiments, X₃ is I, F, S, or T. In any ofthe aforementioned embodiments, X₄ is E, V, or L. In any of theaforementioned embodiments, X₅ is K or R. In any of the aforementionedembodiments, X₆ is E or Q. In any of the aforementioned embodiments, X₇is H, P, or R. In any of the aforementioned embodiments, X₈ is L, T, Sor G. In any of the aforementioned embodiments, X₉ is A. In any of theaforementioned embodiments, X₁₀ is V or I.

In some embodiments, the polypeptide comprises a SIRPα D1 domain thatcomprises at least 85% sequence identity (e.g., at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to SEQ ID NO: 49, wherein each of X₁, X₂, X₃, X₄, X₅, X₆, X₇,X₈, X₉, and X₁₀ are not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of any one of SEQ IDNO: 1. In some embodiments, the polypeptide of this aspect of thedisclosure includes no more than seven amino acid substitutions relativeto the wild-type SIRPα D1 domain having the sequence of any one of SEQID NO: 1.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of any one of SEQ ID NO: 1. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRPα D1 domain having the sequence of any one of SEQID NO: 1. In some embodiments, the polypeptide binds CD47 with at least1000-fold greater binding affinity than the wild-type SIRPα D1 domainhaving the sequence of any one of SEQ ID NO: 1. In some embodiments, aSIRPα D1 domain variant polypeptide or fragment thereof binds to CD47with a K_(D) less than 1×10⁻⁸ M, less than 5×10⁻⁹ M, less than 1×10⁻⁹ M,less 5×10⁻¹ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. In someembodiments, a SIRPα D1 domain variant polypeptide or fragment thereofbinds to CD47 with a K_(D) between about 500 nM and 100 nM, betweenabout 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM,between about 500 pM and 100 pM, between about 100 pM and 50 pM, orbetween about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRPα D1 domain variant having a sequence of:

EEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPIQWFRGAGPARX₄LIYNQX₅X₆GX₇FPRVTTVSEX₈TKRENMDFSISISX₉ITPADAGTYYCX₁₀KFRKGSPDTEFKSGAGTELSVRAKPS, (SEQ IDNO: 50), wherein X₁ is V or I; X₂ is V or I; X₃ is I or F; X₄ is E or V;X₅ is K or R; X₆ is E or Q; X₇ is H or P; X₈ is S or T; X₉ is N or A;and X₁₀ V or I; and wherein the variant comprises at least one aminoacid substitution relative to a wild-type SIRPα D1 domain having thesequence of any one of SEQ ID NO: 2.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 50, wherein X₁ is V or I. In any of the aforementioned embodimentsin this aspect of the disclosure, X₂ is V or I. In any of theaforementioned embodiments, X₃ is I or F. In any of the aforementionedembodiments, X₄ is E or V. In any of the aforementioned embodiments, X₅is K or R. In any of the aforementioned embodiments, X₆ is E or Q. Inany of the aforementioned embodiments, X₇ is H or P. In any of theaforementioned embodiments, X₈ is S or R. In any of the aforementionedembodiments, X₉ is N or A. In any of the aforementioned embodiments, X₁₀is V or I.

In some embodiments, the polypeptide comprises a SIRPα D1 domain thatcomprises at least 85% sequence identity (e.g., at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to SEQ ID NO: 50, wherein each of X₁, X₂, X₃, X₄, X₅, X₆, X₇,X₈, X₉, and X₁₀ is not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of any one of SEQ ID NO:2. In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than seven amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of any one of SEQ ID NO:2.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of any one of SEQ ID NO: 2. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRPα D1 domain having the sequence of any one of SEQID NO: 2. In some embodiments, the polypeptide binds CD47 with at least1000-fold greater binding affinity than the wild-type SIRPα D1 domainhaving the sequence of any one of SEQ ID NO: 2. In some embodiments, aSIRPα D1 domain variant polypeptide or fragment thereof binds to CD47with a K_(D) less than 1×10⁻⁸ M, less than 5×10⁻⁹ M, less than 1×10⁻⁹ M,less 5×10⁻¹ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. In someembodiments, a SIRPα D1 domain variant polypeptide or fragment thereofbinds to CD47 with a K_(D) between about 500 nM and 100 nM, betweenabout 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM,between about 500 pM and 100 pM, between about 100 pM and 50 pM, orbetween about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRPα D1 domain variant having a sequence of:

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅EGX₆FPRVTTVSDX₇TKRNNMDFSIRIGX₈ITPADAGTYYCX₉KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ IDNO: 221), wherein X₁ is V or I; X₂ is A or I; X₃ is I or F; X₄ is E orV; X₅ is K or R; X₆ is H or P; X₇ is L or T; X₈ is N or A; and X₉ is Vor I; and wherein the variant comprises at least one amino acidsubstitution relative to a wild-type SIRPα D1 domain having the sequenceof any one of SEQ ID NO: 1.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 51, wherein X₁ is V or I. In any of the aforementioned embodimentsin this aspect of the disclosure, X₂ is A or I. In any of theaforementioned embodiments, X₃ is I or F. In any of the aforementionedembodiments, X₄ is E or V. In any of the aforementioned embodiments, X₅is K or R. In any of the aforementioned embodiments, X₆ is H or P. Inany of the aforementioned embodiments, X₇ is L or T. In any of theaforementioned embodiments, X₈ is N or A. In any of the aforementionedembodiments, X₉ is V or I. In some embodiments, X₄ is not V.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 51, wherein X₈ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₈ is A and X₁ is V or I. In any of theaforementioned embodiments in this aspect of the disclosure, X₈ is A andX₂ is A or I. In any of the aforementioned embodiments, X₈ is A and X₃is I or F. In any of the aforementioned embodiments, X₈ is A and X₄ is Eor V. In some embodiments, X₄ is not V. In any of the aforementionedembodiments, X₈ is A and X₅ is K or R. In any of the aforementionedembodiments, X₈ is A and X₆ is H or P. In any of the aforementionedembodiments, X₈ is A and X₇ is A or V. In any of the aforementionedembodiments, X₈ is A and X₉ is V or I.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 51, wherein X₈ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₈ is A and X₁ is I. In any of theaforementioned embodiments in this aspect of the disclosure, X₈ is A andX₂ is I. In any of the aforementioned embodiments, X₈ is A and X₃ is F.In any of the aforementioned embodiments, X₈ is A and X₄ is V. In any ofthe aforementioned embodiments, X₈ is A and X₅ is R. In any of theaforementioned embodiments, X₈ is A and X₆ is P. In any of theaforementioned embodiments, X₈ is A and X₇ is T. In any of theaforementioned embodiments, X₈ is A and X₉ is I.

In some embodiments, the polypeptide comprises a SIRPα D1 domain variantthat comprises at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to SEQ ID NO: 51, wherein each of X₁, X₂, X₃, X₄, X₅,X₆, X₇, X₈, and X₉ is not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosurecomprises no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of any one of SEQ IDNO: 1. In some embodiments, the polypeptide of this aspect of thedisclosure comprises no more than seven amino acid substitutionsrelative to the wild-type SIRPα D1 domain having the sequence of any oneof SEQ ID NO: 1.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of any one of SEQ ID NO: 1. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRPα D1 domain having the sequence of any one of SEQID NOs: 1. In some embodiments, the polypeptide binds CD47 with at least1000-fold greater binding affinity than the wild-type SIRPα D1 domainhaving the sequence of any one of SEQ ID NO: 1. In some embodiments, aSIRPα D1 domain variant polypeptide or fragment thereof binds to CD47with a K_(D) less than 1×10⁻⁸ M, less than 5×10⁻⁹ M, less than 1×10⁻⁹ M,less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. In someembodiments, a SIRPα D1 domain variant polypeptide or fragment thereofbinds to CD47 with a K_(D) between about 500 nM and 100 nM, betweenabout 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM,between about 500 pM and 100 pM, between about 100 pM and 50 pM, orbetween about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRPα D1 domain variant having a sequence of:

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRELIYNQX₄EGX₅FPRVTTVSDX₆TKRNNMDFSIRIGX₇ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ IDNO: 222), wherein X₁ is V, L, or I; X₂ is A, I, or L; X₃ is I, T, S, orF; X₄ is K or R; X₅ is H or P; X₆ is L, T, or G; X₇ is N or A; andwherein the variant comprises at least one amino acid substitutionrelative to a wild-type SIRPα D1 domain having a sequence according toSEQ ID NO: 1.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 222, wherein X₁ is V, L, or I. In any of the aforementionedembodiments in this aspect of the disclosure, X₂ is A, I, or L. In anyof the aforementioned embodiments, X₃ is I, T, S, or F. In any of theaforementioned embodiments, X₄ is K or R. In any of the aforementionedembodiments, X₅ is H or P. In any of the aforementioned embodiments, X₆is L, T, or G. In any of the aforementioned embodiments, X₇ is N or A.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 222, wherein X₁ is V or I. In any of the aforementioned embodimentsin this aspect of the disclosure, X₂ is A or I. In any of theaforementioned embodiments, X₃ is I or F. In any of the aforementionedembodiments, X₄ is K or R. In any of the aforementioned embodiments, X₅is H or P. In any of the aforementioned embodiments, X₆ is L or T. Inany of the aforementioned embodiments, X₇ is N or A.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 222, wherein X₇ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₇ is A and X₁ is V or I. In any of theaforementioned embodiments in this aspect of the disclosure, X₇ is A andX₂ is A or I. In any of the aforementioned embodiments, X₇ is A and X₃is I or F. In any of the aforementioned embodiments, X₇ is A and X₄ is Kor R. In any of the aforementioned embodiments, X₇ is A and X₅ is H orP. In any of the aforementioned embodiments, X₇ is A and X₆ is L or T.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 222, wherein X₇ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₇ is A and X₁ is I. In any of theaforementioned embodiments in this aspect of the disclosure, X₇ is A andX₂ is I. In any of the aforementioned embodiments, X₇ is A and X₃ is F.In any of the aforementioned embodiments, X₇ is A and X₄ is R. In any ofthe aforementioned embodiments, X₇ is A and X₅ is P. In any of theaforementioned embodiments, X₇ is A and X₆ is T.

In some embodiments, the polypeptide comprises a SIRPα D1 domain thatcomprises at least 85% sequence identity (e.g., at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to SEQ ID NO: 222, wherein each of X₁, X₂, X₃, X₄, X₅, X₆, andX₇ is not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of any one of SEQ IDNO: 1. In some embodiments, the polypeptide of this aspect of thedisclosure includes no more than seven amino acid substitutions relativeto the wild-type SIRPα D1 domain having the sequence of any one of SEQID NO: 1.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of any one of SEQ ID NO: 1. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRPα D1 domain having the sequence of any one of SEQID NO: 1. In some embodiments, the polypeptide binds CD47 with at least1000-fold greater binding affinity than the wild-type SIRPα D1 domainhaving the sequence of any one of SEQ ID NO: 1. In some embodiments,fragments include polypeptides of less than 10 amino acids in length,about 10 amino acids in length, about 20 amino acids in length, about 30amino acids in length, about 40 amino acids in length, about 50 aminoacids in length, about 60 amino acids in length, about 70 amino acids inlength, about 80 amino acids in length, about 90 amino acids in length,about 100 amino acids in length, or more than about 100 amino acids inlength. Fragments retain the ability to bind to CD47. Preferably, SIRPαD1 domain variant polypeptides and fragments thereof bind to CD47 with ahigher affinity than a SIRPα polypeptide binds to CD47. For example, insome embodiments, a SIRPα D1 domain variant polypeptide or fragmentthereof binds to CD47 with a K_(D) less than 1×10⁻⁸ M, less than5×10⁻⁹M, less than 1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or lessthan 1×10⁻¹¹ M. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) between about500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nMand 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM,between about 1 nM and 500 pM, between about 500 pM and 100 pM, betweenabout 100 pM and 50 pM, or between about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRPα D1 domain variant having a sequence of:

EEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPIQWFRGAGPARELIYNQX₄EGX₅FPRVTTVSEX₆TKRENMDFSISISX₇ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS (SEQ ID NO:212), wherein X₁ is V, L, or I; X₂ is V, I, or L; X₃ is I, T, S, or F;X₄ is K or R; X₅ is H, P, or R; X₆ is S, T, of G; X₇ is N or A; andwherein the variant comprises at least one amino acid substitutionrelative to a wild-type SIRPα D1 domain having the sequence of any oneof SEQ ID NO: 2.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 212, wherein X₁ is V, L, or I. In any of the aforementionedembodiments in this aspect of the disclosure, X₂ is V, I, or L. In anyof the aforementioned embodiments, X₃ is I, T, S, or F. In any of theaforementioned embodiments, X₄ is K or R. In any of the aforementionedembodiments, X₅ is H or P. In any of the aforementioned embodiments, X₆is S, T, or G. In any of the aforementioned embodiments, X₇ is N or A.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 212, wherein X₁ is V or I. In any of the aforementioned embodimentsin this aspect of the disclosure, X₂ is V or I. In any of theaforementioned embodiments, X₃ is I or F. In any of the aforementionedembodiments, X₄ is K or R. In any of the aforementioned embodiments, X₅is H or P. In any of the aforementioned embodiments, X₆ is S or T. Inany of the aforementioned embodiments, X₇ is N or A.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 212, wherein X₇ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₇ is A and X₁ is V or I. In any of theaforementioned embodiments in this aspect of the disclosure, X₇ is A andX₂ is V or I. In any of the aforementioned embodiments, X₇ is A and X₃is I or F. In any of the aforementioned embodiments, X₇ is A and X₄ is Kor R. In any of the aforementioned embodiments, X₇ is A and X₅ is H orP. In any of the aforementioned embodiments, X₇ is A and X₆ is S or T.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 212, wherein X₇ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₇ is A and X₁ is I. In any of theaforementioned embodiments in this aspect of the disclosure, X₇ is A andX₂ is I. In any of the aforementioned embodiments, X₇ is A and X₃ is F.In any of the aforementioned embodiments, X₇ is A and X₄ is R. In any ofthe aforementioned embodiments, X₇ is A and X₅ is P. In any of theaforementioned embodiments, X₇ is A and X₆ is T.

In some embodiments, the polypeptide comprises a SIRPα D1 domain havingat least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity)to SEQ ID NO: 212, wherein each of X₁, X₂, X₃, X₄, X₅, X₆, and X₇ is nota wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of any one of SEQ ID NO:2. In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than seven amino acid substitutions relative to thewild-type SIRPα D1 domain having the sequence of any one of SEQ ID NO:2.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRPα D1 domain having thesequence of any one of SEQ ID NO: 2. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRPα D1 domain having the sequence of any one of SEQID NO: 2. In some embodiments, the polypeptide binds CD47 with at least1000-fold greater binding affinity than the wild-type SIRPα D1 domainhaving the sequence of any one of SEQ ID NO: 2. In some embodiments,fragments include polypeptides of less than 10 amino acids in length,about 10 amino acids in length, about 20 amino acids in length, about 30amino acids in length, about 40 amino acids in length, about 50 aminoacids in length, about 60 amino acids in length, about 70 amino acids inlength, about 80 amino acids in length, about 90 amino acids in length,about 100 amino acids in length, or more than about 100 amino acids inlength. Fragments retain the ability to bind to CD47. Preferably, SIRPαD1 domain variant polypeptides and fragments thereof bind to CD47 with ahigher affinity than a SIRPα polypeptide binds to CD47. For example, insome embodiments, a SIRPα D1 domain variant polypeptide or fragmentthereof binds to CD47 with a K_(D) less than 1×10⁻⁸ M, less than5×10⁻⁹M, less than 1×10⁻⁹ M, less 5×10⁻¹⁰M, less than 1×10⁻¹⁰ M or lessthan 1×10⁻¹¹ M. In some embodiments, a SIRPα D1 domain variantpolypeptide or fragment thereof binds to CD47 with a K_(D) between about500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nMand 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM,between about 1 nM and 500 pM, between about 500 pM and 100 pM, betweenabout 100 pM and 50 pM, or between about 50 pM and 10 pM.

Described herein, in some embodiments, is a polypeptide comprising aSIRPα D1 domain variant having a sequence according to:

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉X₁₀X₁₁X₁₂ADAGTYYCX₁₃KFRKGSPDDVEFKSGAGTELSV RAKPS(SEQ ID NO: 218), wherein X₁ is V, L, or I; X₂ is A, V, L, or I; X₃ isI, S, T, or F; X₄ is E, L, or V; X₅ is K or R; X₆ is E or Q; X₇ is H, R,or P; X₈ is S, G, L, or T; X₉ is any amino acid; X₁₀ is any amino acid;X₁₁ is any amino acid; X₁₂ is any amino acid; and X₁₃ is V or I; andwherein the SIRPα D1 domain variant comprises at least two amino acidsubstitutions relative to a wild-type SIRPα D1 domain having a sequenceaccording to SEQ ID NO: 1.

In some embodiments, the polypeptide comprises the sequence of SEQ IDNO: 212, wherein X₁, wherein X₉ is A. In any of the aforementionedembodiments in this aspect of the disclosure, X₉ is N. In any of theaforementioned embodiments in this aspect of the disclosure X₁₀ is I. Inany of the aforementioned embodiments in this aspect of the disclosureX₉ is N and X₁₀ is P. In any of the aforementioned embodiments in thisaspect of the disclosure X₉ is N and X₁₁ is any amino acid other than S,T, or C. In any of the aforementioned embodiments in this aspect of thedisclosure X₁₁ is T. In any of the aforementioned embodiments in thisaspect of the disclosure X₁₁ is an amino acid other than T. In any ofthe aforementioned embodiments in this aspect of the disclosure X₁₂ isP. In any of the aforementioned embodiments in this aspect of thedisclosure X₉ is N and X₁₂ is any amino acid other than P.

Described herein, in some embodiments, is a polypeptide comprising aSIRPα D1 domain variant having a sequence according to:EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉ITX₁₀ADAGTYYCX₁₁KFRKGSPDDVEFKSGAGTELSVRA KPS(SEQ ID NO: 219), wherein X₁ is V, L, or I; X₂ is A, V, L, or I; X₃ isI, S, T, or F; X₄ is E, L, or V; X₅ is K or R; X₆ is E or Q; X₇ is H, R,or P; X₈ is S, G, L, or T; X₉ is N; X₁₀ is any amino acid other than P;and X₁₁ is V or I; and wherein the SIRPαD1 domain variant comprises atleast two amino acid substitutions relative to a wild-type SIRPα D1domain having a sequence according to SEQ ID NO: 1.

In another aspect of the disclosure, compositions are disclosed hereinwhich include a SIRPα D1 domain variant polypeptide having the aminoacid sequence of SEQ ID NO: 48, or a fragment thereof. In someembodiments, the SIRPα D1 domain variant polypeptide or fragment thereofbinds to CD47 with a higher affinity compared to the affinity that aSIRPα polypeptide binds to the CD47. In some embodiments, the SIRPα D1domain variant polypeptide binds to CD47 with a K_(D) less than 1×10⁻⁸M,or less than 1×10⁻⁹M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹M. In someembodiments, the above-mentioned SIRPα D1 domain variant polypeptidesare attached or fused to a second polypeptide. In some embodiments, thesecond polypeptide includes, without limitation, an Fc polypeptide, anFc variant or a fragment of the foregoing.

Without limiting the foregoing, in some embodiments, a SIRPα D1 domainvariant polypeptide is selected from any one of SEQ ID NOs: 53-87 and213 shown in Table 6.

TABLE 6 SIRPα Variant Polypeptides SEQ ID NO: Amino Acid Sequence  53EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS  54EEELQVIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARELIYNQRQGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGA GTELSVRAKPS  55EEELQVIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS  56EEELQIIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS  57EEELQIIQPDKSVSVAAGESAILHCTITSLIPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGT ELSVRAKPS  58EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS  59EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQKQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS  60EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQREGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS  61EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGHFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS  62EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAG TELSVRAKPS  63EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG TELSVRAKPS  64EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG TELSVRAKPS  65EEELQVIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGA GTELSVRAKPS  66EEELQVIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG TELSVRAKPS  67EEELQVIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG TELSVRAKPS  68EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGT ELSVRAKPS  69EEELQVIQPDKSVSVAAGESAILHCTITSLIPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG TELSVRAKPS  70EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGT ELSVRAKPS  71EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKS GAGTELSVRAKPS  72EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS  73EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS  74EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS  75EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS  76EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKS GAGTELSVRAKPS  77EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSG AGTELSVRAKPS  78EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSG AGTELSVRAKPS  79EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKS GAGTELSVRAKPS  80EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSG AGTELSVRAKPS  81EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKS GAGTELSVRAKPS  82EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS  83EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS  84EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS  85EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS  86EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSG AGTELSVRAKPS  87EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGEIFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKS GAGTELSVRAKPS 195EEELQIIQPDKSVLVAAGETATLRCTMTSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKS GAGTELSVRAKPS 196EEELQIIQPDKSVLVAAGETATLRCTITSLKPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS 197EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS 198EEELQIIQPDKSVLVAAGETATLRCTITSLYPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS 199EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS 200EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSG AGTELSVRAKPS 201EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGMPDDVEFKS GAGTELSVRAKPS 202EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDVEFKSGA GTELSVRAKPS 203EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSSEPDVEFKS GAGTELSVRAKPS 204EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS 205EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSG AGTELSVRAKPS 206EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSG AGTELSVRAKPS 207EEELQIIQPDKSVLVAAGETATLRCTITSLYPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSG AGTELSVRAKPS 208EEELQIIQPDKSVLVAAGETATLRCTITSLYPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSG AGTELSVRAKPS 209EEELQIIQPDKSVLVAAGETATLRCTITSLYPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSG AGTELSVRAKPS 210EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSG AGTELSVRAKPS 213EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITVADAGTYYCVKFRKGSPDDVEFKS GAGTELSVRAKPS

In some embodiments, the polypeptide comprises a SIRPα D1 domain variantthat has at least 85% sequence identity (e.g., at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to any variant provided in Table 6.

In some embodiments, the polypeptide comprises a SIRPα D1 domain thathas at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 (SEQ ID NO: 223)%, or100% sequence identity) to SEQ ID NOs: 80, 81, or 85 in Table 6.

Fc Domain Variants and Fusion Polypeptides Comprising Same

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRPα D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRPα D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRPα D1 domain at a residueselected from the group consisting of: residue 6, residue 27, residue31, residue 47, residue 53, residue 54, residue 56, residue 66, andresidue 92.

Also disclosed herein, in some embodiments, are Fc domain variantdimers, wherein the Fc domain variant dimer comprises two Fc domainvariants, wherein each Fc domain variant independently is selected from(i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A,and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S,P331S and N297A; or (iii) a human IgG4 Fc region comprising mutationsS228P, E233P, F234V, L235A, delG236, and N297A.

Antibodies that target cell surface antigens can triggerimmunostimulatory and effector functions that are associated with Fcreceptor (FcR) engagement on immune cells. There are a number of Fcreceptors that are specific for particular classes of antibodies,including IgG (gamma receptors), IgE (eta receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of the Fc region to Fcreceptors on cell surfaces can trigger a number of biological responsesincluding phagocytosis of antibody-coated particles (antibody-dependentcell-mediated phagocytosis, or ADCP), clearance of immune complexes,lysis of antibody-coated cells by killer cells (antibody-dependentcell-mediated cytotoxicity, or ADCC) and, release of inflammatorymediators, placental transfer, and control of immunoglobulin production.Additionally, binding of the C1 component of complement to antibodiescan activate the complement system. Activation of complement can beimportant for the lysis of cellular pathogens. However, the activationof complement can also stimulate the inflammatory response and can alsobe involved in autoimmune hypersensitivity or other immunologicaldisorders. Variant Fc regions with reduced or ablated ability to bindcertain Fc receptors are useful for developing therapeutic antibodiesand Fc-fusion polypeptide constructs which act by targeting, activating,or neutralizing ligand functions while not damaging or destroying localcells or tissues.

In some embodiments, a SIRPα D1 polypeptide construct comprises anon-naturally occurring SIRPα D1 domain variant linked to an Fc domainvariant which forms an Fc domain having ablated or reduced effectorfunction.

In some embodiments, a Fc domain variant refers to a polypeptide chainthat includes second and third antibody constant domains (e.g., CH2 andCH3). In some embodiments, an Fc domain variant also includes a hingedomain. In some embodiments, the Fc domain variant is of anyimmunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, and IgD.Additionally, in some embodiments, an Fc domain variant is of any IgGsubtype (e.g., IgG, IgG2, IgG2a, IgG2b, IgG2c, IgG3, and IgG4). In someembodiments, an Fc domain variant comprises as many as ten amino acidmodifications (e.g., insertions, deletions and/or substitutions)relative to a wild-type Fc domain monomer sequence (e.g., 1-10, 1-8,1-6, 1-4 amino acid substitutions, additions or insertions, deletions,or combinations thereof) that alter the interaction between an Fc domainand an Fc receptor.

As used herein, the term “Fc domain dimer” refers to a dimer of two Fcdomains. In a wild-type Fc domain dimer, two wild-type Fc domainsdimerize by the interaction between the two CH3 antibody constantdomains, as well as one or more disulfide bonds that form between thehinge domains of the two dimerized Fc domains.

As used herein, the term “Fc domain dimer variant” comprises at leastone Fc domain variant. In some embodiments, an Fc domain dimer variantcomprises Fc domain variants that are mutated to lack effectorfunctions, for example a “dead Fc domain dimer variant.” In someembodiments, each of the Fc domains in an Fc domain dimer variantincludes amino acid substitutions in the CH2 antibody constant domain toreduce the interaction or binding between the Fc domain dimer variantand an Fc receptor, such as an Fcγ receptor (FcγR), an Fcα receptor(FcαR), or an Fcε (FcεR).

In some embodiments, a SIRPα D1 domain variant (e.g., any of thevariants described in Tables 2, 5, and 6) is fused to an Fc domainvariant of an immunoglobulin or a fragment of an Fc domain variant. Insome embodiments, an Fc domain variant of an immunoglobulin or afragment of an Fc domain variant is capable of forming an Fc domaindimer with another Fc domain variant. In some embodiments, an Fc domainvariant of an immunoglobulin or a fragment of an Fc domain variant isnot capable of forming an Fc domain dimer with another Fc domainvariant. In some embodiments, an Fc domain variant or a fragment of anFc domain variant is fused to a polypeptide of the disclosure toincrease serum half-life of the polypeptide. In some embodiments, an Fcdomain variant or a fragment of an Fc domain variant fused to apolypeptide of the disclosure dimerizes with a second Fc domain variantto form an Fc domain dimer variant which binds an Fc receptor, oralternatively, an Fc domain variant binds to an Fc receptor. In someembodiments, an Fc domain variant or a fragment of the Fc domain variantfused to a polypeptide to increase serum half-life of the polypeptidedoes not induce any immune system-related response.

In some embodiments, a SIRPα polypeptide or construct provided hereinincludes a SIRPα D1 domain or variant thereof joined to a first Fcdomain variant and an antibody variable domain joined to a second Fcdomain variant, in which the first and second Fc domain variants combineto form an Fc domain dimer variant (e.g., a heterodimeric Fc domaindimer variant). An Fc domain dimer is the protein structure that isfound at the C-terminus of an immunoglobulin. An Fc domain dimerincludes two Fc domains that are dimerized by the interaction betweenthe CH3 antibody constant domains. A wild-type Fc domain dimer forms theminimum structure that binds to an Fc receptor, e.g., FcγRI, FcγRIIa,FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV.

The Fc domain dimer is not involved directly in binding an antibody toits target, but can be involved in various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.In some embodiments, the Fc domain in a SIRPα polypeptide or constructof the disclosure comprises amino acid substitutions, additions orinsertions, deletions, or any combinations thereof that lead todecreased effector function such as decreased antibody-dependentcell-mediated cytotoxicity (ADCC), decreased complement-dependentcytolysis (CDC), decreased antibody-dependent cell-mediated phagocytosis(ADCP), or any combinations thereof. In some embodiments, the SIRPαpolypeptides or constructs of the disclosure are characterized bydecreased binding (e.g., minimal binding or absence of binding) to ahuman Fc receptor and decreased binding (e.g., minimal binding orabsence of binding) to complement protein C1q. In some embodiments, theSIRPα constructs of the disclosure are characterized by decreasedbinding (e.g., minimal binding or absence of binding) to human FcγRI,FcγRIIA, FcγRIIB, FcγRIIIB, or any combinations thereof, and C1q. Toalter or reduce an antibody-dependent effector function, such as ADCC,CDC, ADCP, or any combinations thereof, in some embodiments, the Fcdomains in SIRPα constructs of the disclosure are of the IgG class andcomprise one or more amino acid substitutions at E233, L234, L235, G236,G237, D265, D270, N297, E318, K320, K322, A327, A330, P331, or P329(numbering according to the EU index of Kabat (Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991))).

In some embodiments, polypeptide constructs comprising a non-native Fcregion described herein exhibit reduced or ablated binding to at leastone of Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 as compared toa polypeptide construct comprising a native Fc region. In some cases,the polypeptide constructs described herein exhibit reduced or ablatedbinding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors.

CDC refers to a form of cytotoxicity in which the complement cascade isactivated by the complement component C1q binding to antibody Fcdomains. In some embodiments, polypeptide constructs comprising anon-native Fc region described herein exhibit at least a 5%, 10%, 15%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in C1qbinding compared to a polypeptide construct comprising a wild-type Fcregion. In some cases, polypeptide constructs comprising a non-native Fcregion as described herein exhibit reduced CDC as compared to apolypeptide construct comprising a wild-type Fc region. In someembodiments, polypeptide constructs comprising a non-native Fc region asdescribed herein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or greater reduction in CDC compared to a polypeptideconstruct comprising a wild-type Fc region. In some cases, polypeptideconstructs comprising a non-natural Fc domain variants or Fc domaindimer variants as described herein exhibit negligible CDC as compared toa polypeptide construct comprising a wild-type Fc region.

In some embodiments, the Fc domain variants or Fc domain dimer variantsdescribed herein are minimally glycosylated or have reducedglycosylation relative to a wild-type sequence. In some embodiments,deglycosylation is accomplished with a mutation of N297A, or by mutatingN297 to any amino acid which is not N. In some embodiments,deglycosylation is accomplished by disrupting the motifN-Xaa1-Xaa2-Xaa3, wherein N=asparagine; Xaa1=any amino acid except P(proline); Xaa2=T (threonine), S (serine) or C (cysteine); and Xaa3=anyamino acid except P (proline). In one embodiment, the N-Xaa1-Xaa2-Xaa3motif refers to residues 297-300 as designated according to Kabat etal., 1991. In some embodiments, a mutation to any one or more of N,Xaa1, Xaa2, or Xaa3 results in deglycosylation of the Fc domain variantor Fc domain dimer variant.

In some embodiments, variants of antibody IgG constant regions (e.g., Fcdomain variants or Fc domain dimer variants) possess a reduced capacityto specifically bind Fcγ receptors or have a reduced capacity to inducephagocytosis. In some embodiments, variants of antibody IgG constantregions (e.g., Fc domain variants or Fc domain dimer variants) possess areduced capacity to specifically bind Fcγ receptors and have a reducedcapacity to induce phagocytosis. For example, in some embodiments, an Fcdomain variant is mutated to lack effector functions, typical of a“dead” Fc domain variant. For example, in some embodiments, an Fc domainvariant includes specific amino acid substitutions that are known tominimize the interaction between the Fc domain dimer and an Fcγreceptor. In some embodiments, an Fc domain variant is from an IgG1antibody and includes one or more of amino acid substitutions L234A,L235A, G237A, and N297A (as designated according to the EU numberingsystem per Kabat et al., 1991). In some embodiments, one or moreadditional mutations are included in such IgG1 Fc domain variant.Non-limiting examples of such additional mutations for human IgG1 Fedomain variants include E318A and K322A. In some instances, a human IgG1Fc domain variant has up to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewermutations in total as compared to wild-type human IgG1 sequence. In someembodiments, one or more additional deletions are included in such IgG1Fc domain variant. For example, in some embodiments, the C-terminallysine of the Fc domain IgG1 heavy chain constant region provided in SEQID NO: 88 in Table 7 is deleted, for example to increase the homogeneityof the polypeptide when the polypeptide is produced in bacterial ormammalian cells. In some instances, a human IgG1 Fc domain variant hasup to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewer deletions in total ascompared to wild-type human IgG1 sequence (see, e.g., SEQ ID NO: 161below). In some embodiments, a IgG1 Fc domain variant has a sequenceaccording to any one of SEQ ID NO: 135, SEQ ID NO: 136 or SEQ ID NO:137.

SEQ ID NO: 161: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

In some embodiments, an Fc domain variant is from an IgG2 or IgG4antibody and includes amino acid substitutions A330S, P331S, or bothA330S and P331S. The aforementioned amino acid positions are definedaccording to Kabat, et al. (1991). The Kabat numbering of amino acidresidues can be determined for a given antibody by alignment at regionsof homology of the sequence of the antibody with a “standard” Kabatnumbered sequence. In some embodiments, the Fc domain variant comprisesa human IgG2 Fc domain sequence comprising one or more of A330S, P331Sand N297A amino acid substitutions (as designated according to the EUnumbering system per Kabat, et al. (1991). In some embodiments, one ormore additional mutations are included in such IgG2 Fc domain variants.Non-limiting examples of such additional mutations for human IgG2 Fcdomain variant include V234A, G237A, P238S, V309L and H268A (asdesignated according to the EU numbering system per Kabat et al.(1991)). In some instances, a human IgG2 Fc domain variant has up to 12,11, 10, 9, 8, 7, 6, 5, 4, 3 or fewer mutations in total as compared towild-type human IgG2 sequence. In some embodiments, one or moreadditional deletions are included in such IgG2 Fc domain variant. Forexample, in some embodiments, the C-terminal lysine of the Fc domainIgG2 heavy chain constant region provided in SEQ ID NO: 89 in Table 7 isdeleted, for example to increase the homogeneity of the polypeptide whenthe polypeptide is produced in bacterial or mammalian cells. In someinstances, a human IgG2 Fc domain variant has up to 12, 11, 10, 9, 8, 7,6, 5 or 4 or fewer deletions in total as compared to wild-type humanIgG2 sequence (see, e.g., SEQ ID NO: 162 below).

SEQ ID NO: 162: ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSMVQQGNVFSCSVMHEALHNHYTQKSLSLSPG

When the Fc domain variant is an IgG4 Fc domain variant, in someembodiments, such Fc domain variant comprises a S228P mutation (asdesignated according to Kabat, et al. (1991)). In some instances, ahuman IgG4 Fc domain variant has up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 mutation(s) in total as compared to wild-type human IgG4sequence. In some embodiments, the Fc domain variant comprises a humanIgG4 Fc sequence comprising one or more of S228P, E233P, F234V, L235A,and delG236 amino acid substitutions (as designated according to the EUnumbering system per Kabat, et al. (1991). In some embodiments, the Fcdomain variant comprises a human IgG4 Fc sequence comprising one or moreof S228P, E233P, F234V, L235A, deG236, and N297A amino acidsubstitutions (as designated according to the EU numbering system perKabat, et al. (1991).

In some embodiments, the Fc domain variant includes at least one of themutations L234A, L235A, G237A or N297A of an IgG1 Fc region or at leastone of the mutations A330S, P331S or N297A of an IgG2 Fc region. In someembodiments, the Fc domain variant includes at least two of themutations L234A, L235A, G237A or N297A of an IgG1 Fc region or at leasttwo of the mutations A330S, P331S or N297A of an IgG2 Fc region. In someembodiments, the Fc domain variant includes at least three of themutations L234A, L235A, G237A or N297A of an IgG1 Fc region or consistsof the mutations A330S, P331S and N297A of an IgG2 Fc region. In someembodiments, the Fc domain variant consists of the mutations L234A,L235A, G237A and N297A.

In some embodiments, the Fc domain variant exhibits reduced binding toan Fc receptor of the subject compared to the wild-type human IgG Fcregion. In some embodiments, the Fc domain variant exhibits ablatedbinding to an Fc receptor of the subject compared to the wild-type humanIgG Fc region. In some embodiments, the Fc domain variant exhibits areduction of phagocytosis compared to the wild-type human IgG Fc region.In some embodiments, the Fc domain variant exhibits ablated phagocytosiscompared to the wild-type human IgG Fc region. SEQ ID NO: 88 and SEQ IDNO: 89 provide amino acid sequences of Fc domain IgG1 and IgG2 heavychain constant regions. In some embodiments, an Fc domain variant is anyvariant of SEQ ID NOs: 90-95 as shown in Table 7.

TABLE 7 Amino Acid Sequences of Fc Domain Variants SEQ ID NO:Amino Acid Sequence 88EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 89STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDEIKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 90DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 91DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 92VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK93 VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 94ERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 95ERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Antibody-dependent cell-mediated cytotoxicity, which is also referred toherein as ADCC, refers to a form of cytotoxicity in which secreted Igbound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g.,Natural Killer (NK) cells and neutrophils) enabling these cytotoxiceffector cells to bind specifically to an antigen-bearing target celland subsequently kill the target cell. Antibody-dependent cell-mediatedphagocytosis, which is also referred to herein as ADCP, refers to a formof cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain phagocytic cells (e.g., macrophages) enabling thesephagocytic effector cells to bind specifically to an antigen-bearingtarget cell and subsequently engulf and digest the target cell.Ligand-specific high-affinity IgG antibodies directed to the surface oftarget cells can stimulate the cytotoxic or phagocytic cells and can beused for such killing. In some embodiments, polypeptide constructscomprising an Fc domain variant or Fc domain dimer variant as describedherein exhibit reduced ADCC or ADCP as compared to a polypeptideconstruct comprising a wild-type Fc region. In some embodiments,polypeptide constructs comprising an Fc domain variant or Fc domaindimer variant as described herein exhibit at least a 5%, 10%, 15%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in ADCC or ADCPcompared to a polypeptide construct comprising a wild-type Fc region. Insome embodiments, polypeptide constructs comprising an Fc domain variantor Fc domain dimer variant as described herein exhibit ablated ADCC orADCP as compared to a polypeptide construct comprising a wild-type Fcregion.

Complement-directed cytotoxicity, which is also referred to herein asCDC, refers to a form of cytotoxicity in which the complement cascade isactivated by the complement component C1q binding to antibody Fcdomains. In some embodiments, polypeptide constructs comprising an Fcdomain variant or Fc domain dimer variant as described herein exhibit atleast a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greaterreduction in C1q binding compared to a polypeptide construct comprisinga wild-type Fc region. In some cases, polypeptide constructs comprisingan Fe domain variant or Fe domain dimer variant as described hereinexhibit reduced CDC as compared to a polypeptide construct comprising awild-type Fc region. In some embodiments, polypeptide constructscomprising an Fc domain variant or Fc domain dimer variant as describedherein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or greater reduction in CDC compared to a polypeptide constructcomprising a wild-type Fc region. In some cases, polypeptide constructscomprising an Fc domain variant or Fc domain dimer variant as describedherein exhibit negligible CDC as compared to a polypeptide constructcomprising a wild-type Fc region.

Fc domain variants or Fc domain dimer variants herein include those thatexhibit reduced binding to an Fcγ receptor compared to the wild-typehuman IgG Fc region. For example, in some embodiments, an Fc domainvariant or Fc domain dimer variant exhibits binding to an Fcγ receptorthat is less than the binding exhibited by a wild-type human IgG Fcregion to an Fcγ receptor, as described in the Examples. In someinstances, an Fc domain variant or Fc domain dimer variant has reducedbinding to an Fcγ receptor by a factor of 10%, 20% 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (fully ablated effectorfunction). In some embodiments, the reduced binding is for any one ormore Fcγ receptor, e.g., CD16a, CD32a, CD32b, CD32c, or CD64.

In some instances, the Fc domain variants or Fc domain dimer variantsdisclosed herein exhibit a reduction of phagocytosis compared to itswild-type human IgG Fc region. Such Fc domain variants or Fc domaindimer variants exhibit a reduction in phagocytosis compared to itswild-type human IgG Fc region, wherein the reduction of phagocytosisactivity is e.g., by a factor of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99% or 100%. In some instances, an Fc domainvariant or Fc domain dimer variant exhibits ablated phagocytosiscompared to its wild-type human IgG Fc region.

In some embodiments, the Fc domain variants or Fc domain dimer variantsdisclosed herein are coupled to one or more fusion partners. In somecases the fusion partner is a therapeutic moiety. In some cases, thefusion partner is selected to enable targeting of an expressed protein,purification, screening, display, and the like. In some embodiments, thefusion partner also affects the degree of binding to Fc receptors or thedegree of phagocytosis reduction. As described herein, in someembodiments, when an Fc domain variant or Fc domain dimer variant iscoupled to a fusion partner, it forms a polypeptide construct asdescribed below.

In some embodiments, fusion partners are linked to the Fe domain variantor Fe domain dimer variant sequence via a linker sequence. In someembodiments, the linker sequence generally comprises a small number ofamino acids, such as less than ten amino acids, although longer linkersare also utilized. In some cases, the linker has a length less than 10,9, 8, 7, 6, or 5 amino acids or shorter. In some cases, the linker has alength of at least 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35 amino acidsor longer. Optionally, in some embodiments, a cleavable linker isemployed.

In some embodiments, a fusion partner is a targeting or signal sequencethat directs an Fc domain variant or Fc domain dimer variant protein andany associated fusion partners to a desired cellular location or to theextracellular media. In some embodiments, certain signaling sequencestarget a protein to be either secreted into the growth media, or intothe periplasmic space, located between the inner and outer membrane ofthe cell. In some embodiments, a fusion partner is a sequence thatencodes a peptide or protein that enables purification or screening.Such fusion partners include, but are not limited to, polyhistidine tags(His-tags) (for example His6 (SEQ ID NO: 223) and His10 (SEQ ID NO:224)) or other tags for use with Immobilized Metal AffinityChromatography (IMAC) systems (e.g., Ni+2 affinity columns), GSTfusions, MBP fusions, Strep-tag, the BSP biotinylation target sequenceof the bacterial enzyme BirA, and epitope tags which are targeted byantibodies (for example c-myc tags, flag-tags, and the like).

In some embodiments, such tags are useful for purification, forscreening, or both. For example, in some embodiments, an Fc domainvariant or Fc domain dimer variant is purified using a His-tag byimmobilizing it to a Ni+2 affinity column, and then after purificationthe same His-tag is used to immobilize the antibody to a Ni+2 coatedplate to perform an ELISA or other binding assay as described elsewhereherein. In some embodiments, a fusion partner enables the use of aselection method to screen Fc domain variants or Fc domain dimervariants as described herein.

Various fusion partners that enable a variety of selection methods areavailable. For example, by fusing the members of an Fc domain variant orFc domain dimer variant library to the gene III protein, phage displaycan be employed. In some embodiments, fusion partners Fc domain variantsor Fc domain dimer variants to be labeled. Alternatively, in someembodiments, a fusion partner binds to a specific sequence on theexpression vector, enabling the fusion partner and associated Fc domainvariant or Fc domain dimer variant to be linked covalently ornoncovalently with the nucleic acid that encodes them.

In some embodiments, when a fusion partner is a therapeutic moiety, thetherapeutic moiety is, e.g., a peptide, a protein, an antibody, a siRNA,or a small molecule. Non-limiting examples of therapeutic antibodiesthat are coupled to the Fc domain variants or Fc domain dimer variantsof the present disclosure include, but are not limited to antibodiesthat recognize CD47. Non-limiting examples of therapeutic polypeptidesthat are coupled to the Fc domain variants or Fc domain dimer variantsof the present disclosure include, but are not limited to, CD47 bindingpolypeptides, including SIRPα polypeptides. In such instances, the CD47binding polypeptide is attached or fused to an Fc domain variant or Fcdomain dimer variant of the disclosure. Examples of CD47 bindingpolypeptides include, but are not limited to, anti-CD47 antibodies orfragments thereof, and ligands of CD47 such as SIRPα or a fragmentthereof. Additional examples of CD47 binding polypeptides include, butare not limited to naturally-occurring forms of SIRPα as well as mutantsthereof.

In some embodiments, disclosed herein is a polypeptide comprising an Fcdomain dimer variant, wherein the Fc domain dimer variant comprises twoFc domain variants, wherein each Fc domain variant independently isselected from (i) a human IgG1 Fc region consisting of mutations L234A,L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting ofmutations A330S, P331S and N297A; or (iii) a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. Insome embodiments, the Fc domain variants are identical (i.e.,homodimer). In some embodiments, the Fc domain variants are different(i.e., heterodimer). In some embodiments, at least one of the Fc domainvariant in an Fc domain dimer is a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A. In some embodiments, at leastone of the Fc domain variants in an Fc domain dimer is a human IgG2 Fcregion consisting of mutations A330S, P331S and N297A. In someembodiments, the Fc domain dimer variant exhibits ablated or reducedbinding to an Fcγ receptor compared to the wild-type version of thehuman IgG Fc region. In some embodiments, the Fc domain dimer variantexhibits ablated or reduced binding to CD16a, CD32a, CD32b, CD32c, andCD64 Fcγ receptors compared to the wild-type version of the human IgG Fcregion. In some embodiments, the Fc domain dimer variant exhibitsablated or reduced binding to C1q compared to the wild-type version ofthe human IgG Fc fusion. In some embodiments, at least one of the Fcdomain variants in an Fc domain dimer variant is a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. Insome embodiments, the Fc domain dimer variant exhibits ablated orreduced binding to an Fcγ receptor compared to the wild-type human IgG4Fc region. In some embodiments, the Fc domain dimer variant exhibitsablated or reduced binding to CD16a and CD32b Fcγ receptors compared tothe wild-type version of its human IgG4 Fc region. In some embodiments,the Fc domain dimer variant binds to an Fcγ receptor with a K_(D)greater than about 5×10⁻⁶ M.

In some embodiments, the Fc domain dimer variant further comprises aCD47 binding polypeptide. In some embodiments, the Fc domain dimervariant exhibits ablated or reduced binding to an Fcγ receptor comparedto a wild-type version of a human IgG Fc region. In some embodiments,the CD47 binding polypeptide does not cause acute anemia in rodents andnon-human primates. In some embodiments, the CD47 binding polypeptidedoes not cause acute anemia in humans.

In some embodiments, the CD47 binding polypeptide is a signal-regulatoryprotein α (SIRP-α) polypeptide or a fragment thereof. In someembodiments, the SIRPα polypeptide comprises a SIRPα D1 domain variantcomprising the amino acid sequence,EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅EGX₆FPRVTTVSDX₇TKRNNMDFSIRIGX₈ITPADAGTYYCX₉KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ IDNO: 221), wherein X₁ is V or I; X₂ is A or I; X₃ is I or F; X₄ is E orV; X₅ is K or R; X₆ is H or P; X₇ is L or T; X₈ is any amino acid otherthan N; and X₉ is V or I. In some embodiments, the SIRPα polypeptidecomprises a SIRPα D1 domain variant wherein X₁ is V or I; X₂ is A or I;X₃ is I or F; X₄ is E; X₅ is K or R; X₆ is H or P; X₇ is L or T; X₈ isnot N; and X₉ is V.

In some embodiments, disclosed herein, is a polypeptide comprising: aSIRPα D1 domain variant, wherein the SIRPα D1 domain variant is anon-naturally occurring high affinity SIRPα D1 domain, wherein the SIRPαD1 domain variant binds to human CD47 with an affinity that is at least10-fold greater than the affinity of a naturally occurring D1 domain;and an Fc domain variant, wherein the Fc domain variant is linked to asecond polypeptide comprising a second Fc domain variant to form an Fcdomain dimer variant, wherein the Fc domain dimer variant has ablated orreduced effector function. In some embodiments, the non-naturallyoccurring high affinity SIRPα D1 domain comprises an amino acid mutationat residue 80.

In some embodiments, disclosed herein, is a SIRPα D1 domain variant,wherein the SIRPα D1 domain variant binds CD47 from a first species witha KD less than 250 nM; and wherein the SIRPα D1 domain variant bindsCD47 from a second species with a KD less than 250 nM; and the KD forCD47 from the first species and the KD for CD47 from the second speciesare within 100 fold of each other; wherein the first species and thesecond species are selected from the group consisting of human, rodent,and non-human primate. In some embodiments, the SIRPα D1 domain variantbinds CD47 from at least 3 different species. In some embodiments, thenon-human primate is cynomolgus monkey.

In some embodiments, disclosed herein, is a polypeptide comprising (a) aSIRPα D1 domain that binds human CD47 with a K_(D) less than 250 nM; and(b) an Fc domain or variant thereof linked to the N-terminus or theC-terminus of the SIRPα D1 domain, wherein the polypeptide does notcause acute anemia in rodents and non-human primates. In someembodiments, the polypeptide is a non-naturally occurring variant of ahuman SIRP-α. In some embodiments, administration of the polypeptide invivo results in hemoglobin reduction by less than 50% during the firstweek after administration. In some embodiments, administration of thepolypeptide in humans results in hemoglobin reduction by less than 50%during the first week after administration. In some embodiments, thepolypeptide further comprises at least one Fc domain dimer variant,wherein the Fc domain dimer variant comprises an Fc domain variantselected from (i) a human IgG1 Fc region consisting of mutations L234A,L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting ofmutations A330S, P331S and N297A; or (iii) a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. Insome embodiments, the Fc domain variant is a human IgG1 Fc regionconsisting of mutations L234A, L235A, G237A, and N297A. In someembodiments, the Fc domain variant is a human IgG2 Fc region consistingof mutations A330S, P331S and N297A.

The SIRPα constructs of the disclosure include a SIRPα domain or variantthereof that has its C-terminus joined to the N-terminus of an Fc domainor variant thereof by way of a linker using conventional genetic orchemical means, e.g., chemical conjugation. In some embodiments, alinker (e.g., a spacer) is inserted between the polypeptide and the Fcdomain or variant thereof. In some embodiments, a polypeptide of thedisclosure including a SIRPα D1 domain variant is fused to an Fc domainvariant that is incapable of forming a dimer. In some embodiments, apolypeptide of the disclosure is fused to an Fc domain or variantthereof that is capable of forming a dimer, e.g., a heterodimer, withanother Fc domain or variant thereof. In some embodiments, a polypeptideof the invention is fused to an Fc domain or variant thereof and thisfusion protein forms a homodimer. In some embodiments, a polypeptide ofthe disclosure is fused to a first Fc domain or variant thereof and adifferent protein or peptide (e.g., an antibody variable region) isfused to a second Fc domain or variant thereof. In some embodiments, aSIRPα D1 domain or variant thereof is joined to a first Fe domain orvariant thereof and a therapeutic protein (e.g., a cytokine, aninterleukin, an antigen, a steroid, an anti-inflammatory agent, or animmunomodulatory agent) is joined to a second Fc domain or variantthereof. In some embodiments, the first and second Fc domains orvariants thereof form a heterodimer.

Without the limiting the foregoing, in some embodiments, a SIRPα D1domain variant polypeptide (e.g., any of the variants described inTables 2, 5, and 6) is fused to an Fc polypeptide or Fc variantpolypeptide, such as an Fc domain or variant thereof. Examples ofpolypeptides comprising a SIRPα D1 domain variant polypeptide and afused Fc domain variant polypeptide include, but are not limited to, SEQID NOS: 96-137, 214, and 216 shown in Table 8.

TABLE 8 Polypeptides Comprising SIRPα D1 Domain Variants Fused to FcDomain Variants SEQ ID NO: Amino Acid Sequence  96EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  97EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  98EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  99EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 100EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 101EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 102EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 103EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 104EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 105EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 106EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 107EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 108EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 109EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVEINAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 110EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 111EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 112EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 113EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 114EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 115EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 116EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 117EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 118EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 119EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 120EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 121EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 122EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 123EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEIEDPEVKFNWYVDGVEVEINAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 124EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 125EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVEINAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 126EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 127EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 128EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 129EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 130EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGEIFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALEINHYTQKSLSLSLGK 131EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVEINAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALEINHYTQKSLSLSLGK 132EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVEINAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALEINHYTQKSLSLSLGK 133EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALEINHYTQKSLSLSLGK 134EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSAAAPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALEINHYTQKSLSLSPGK 135EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 136EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPG 137EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPG 211EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPG 214EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPG 216EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVEINAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSPGK 217EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSEKTHTCPECPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALIINHYTQKSLSLSPGK

In some embodiments, the polypeptide comprises a SIRPα D1 variant domainthat has at least 85% sequence identity (e.g., at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to any variant provided in Table 8.

In some embodiments, the polypeptide comprises a SIRPα D1 domain variantthat has at least 85% sequence identity (e.g., at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to SEQ ID NOs: 98-104, 107-113, 116-122, or 135-137 in Table8.

In some embodiments, the polypeptide comprises (a) a signal-regulatoryprotein α (SIRP-α) D1 variant, wherein the SIRPα D1 domain variantcomprises the amino acid sequence,EEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃X₂₄ITX₂₅ADAGTYYCX₂₆KX₂₇RKGSPDX₂₈X₂₉EX₃₀KSGAGTELSVRX₃₁KPS (SEQ ID NO: 47), wherein X₁ is E, or G; X₂is L, I, or V; X₃ is V, L, or I; X₄ is S, or F; X₅ is L, or S; X₆ is S,or T; X₇ is A, or V; X₈ is I, or T; X₉ is H, R, or L; X₁₀ is A, V, I, orL; X₁₁ is I, T, S, or F; X₁₂ is A, or G; X₁₃ is E, V, or L; X₁₄ is K, orR; X₁₅ is E, or Q; X₁₆ is H, P, or R; X₁₇ is D, or E; X₁₈ is S, L, T, orG; X₁₉ is K, or R; X₂₀ is E, or N; X₂₁ is S, or P; X₂₂ is S, or R; X₂₃is S, or G; X₂₄ is any amino acid; X₂₅ is any amino acid; X₂₆ is V, orI; X₂₇ is F, L, or V; X₂₈ is D or absent; X₂₉ is T, or V; X₃₀ is F, orV; and X₃₁ is A, or G; and wherein the SIRPα D1 domain variant comprisesat least two amino acid substitutions relative to a wild-type SIRPα Ddomain having a sequence according to any one of SEQ ID NOs: 1 to 10;and (b) an Fc domain dimer variant having two Fc domain variants,wherein each Fc domain variant independently is (i) a human IgG1 Fcregion comprising a N297A mutation; (ii) a human IgG1 Fc regioncomprising L234A, L235A, and G237A mutations; (iii) a human IgG1 Fcregion comprising L234A, L235A, G237A, and N297A mutations; (iv) a humanIgG2 Fc region comprising a N297A mutation; (v) a human IgG2 Fc regioncomprising A330S and P331S mutations; (vi) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations; (vii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations; or(viii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations.

In some embodiments, the polypeptide comprises a SIRPα D1 domain variantwherein the SIRPα D1 domain variant comprises an amino acid sequenceaccording to SEQ ID NO: 47; an Fc domain dimer having two Fc domains,wherein one of the Fc domains is an Fc domain variant comprising a humanIgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations.

Dimerization of Fc Domains

In some embodiments, a SIRPα D1 domain variant polypeptide (e.g., any ofthe variants described in Tables 2, 5, and 6) is fused to a first Fcdomain (e.g., an Fc domain variant) either at the N-terminus or at theC-terminus. In some embodiments, the first Fc domain is a variant thatis incapable of forming an dimer. In some embodiments, the first Fcdomain forms a dimer with a second Fc domain. In some embodiments, thefirst and second Fc domains comprise amino acid substitutions thatpromote heterodimerization between the first and second domain Fcdomains.

In some embodiments, each of the two Fc domains in an Fc domain dimerincludes amino acid substitutions that promote the heterodimerization ofthe two monomers. In some embodiments, a SIRPα construct is formed, forexample, from a first subunit including a SIRPα D1 domain variantpolypeptide fused to a first Fc domain and a second subunit including asecond Fc domain (e.g., without a SIRPα D1 domain variant polypeptide orany other polypeptide). In some embodiments, a construct has a singleSIRPα D1 domain variant polypeptide linked to an Fc domain dimer (e.g.,single arm). In some embodiments, a construct has two SIRPα D1 domainvariant polypeptides linked to an Fc domain dimer (e.g., double arm). Insome embodiments, a SIRPα D1 domain variant having a K_(D) of about 500nM is particularly useful in a double arm construct. In someembodiments, a SIRPα D1 domain variant having a K_(D) of about 50 nM isparticularly useful in a double arm construct. In some embodiments, aSIRPα D1 domain variant having a K_(D) of about 5 nM is useful in adouble arm construct and a single arm construct. In some embodiments, aSIRPα D1 domain variant having a K_(D) of about 500 pM is useful in adouble arm construct and a single arm construct. In some embodiments, aSIRPα D1 domain variant having a K_(D) of about 100 pM is useful in adouble arm construct and a single arm construct. In some embodiments, aSIRPα D1 domain variant having a K_(D) of about 50 pM is useful in adouble arm construct and a single arm construct. In some embodiments, aSIRPα D1 domain variant having a K_(D) of about 10 pM is useful in adouble arm construct and a single arm construct.

In some embodiments, heterodimerization of Fc domains is promoted byintroducing different, but compatible, substitutions in the two Fcdomains, such as “knob-into-hole” residue pairs and charge residuepairs. The knob and hole interaction favors heterodimer formation,whereas the knob-knob and the hole-hole interaction hinder homodimerformation due to steric clash and deletion of favorable interactions. Ahole refers to a void that is created when an original amino acid in aprotein is replaced with a different amino acid having a smallerside-chain volume. A knob refers to a bump that is created when anoriginal amino acid in a protein is replaced with a different amino acidhaving a larger side-chain volume. For example, in some embodiments, anamino acid being replaced is in the CH3 antibody constant domain of anFc domain and involved in the dimerization of two Fc domains. In someembodiments, a hole in one CH3 antibody constant domain is created toaccommodate a knob in another CH3 antibody constant domain, such thatthe knob and hole amino acids act to promote or favor theheterodimerization of the two Fc domains. In some embodiments, a hole inone CH3 antibody constant domain is created to better accommodate anoriginal amino acid in another CH3 antibody constant domain. In someembodiments, a knob in one CH3 antibody constant domain is created toform additional interactions with original amino acids in another CH3antibody constant domain.

In some embodiments, a hole is constructed by replacing amino acidshaving larger side chains such as tyrosine or tryptophan with aminoacids having smaller side chains such as alanine, valine, or threonine,for example a Y407V mutation in the CH3 antibody constant domain.Similarly, in some embodiments, a knob is constructed by replacing aminoacids having smaller side chains with amino acids having larger sidechains, for example a T366W mutation in the CH3 antibody constantdomain. In some embodiments, one Fc domain includes the knob mutationT366W and the other Fc domain includes hole mutations T366S, L358A, andY407V. In some embodiments, a polypeptide of the disclosure including aSIRPα D1 domain variant is fused to an Fc domain including the knobmutation T366W to limit unwanted knob-knob homodimer formation. Examplesof knob-into-hole amino acid pairs are included, without limitation, inTable 9 and examples of knob-into-hole Fc domain variants and SIRPα-Fcfusions are provided in Table 10.

TABLE 9 Knob-into-Hole Amino Acid Pairs First Fc Y407T Y407A F405A T394ST366S T394W T394S T366W Domain L358A Y407T Y407A T394S Y407V Second FcT366Y T366W T394W F405W T366W T366Y T366W F405W Domain F405A F405W Y407A

TABLE 10Exemplary Fc Domain Variants and SIRPα D1 Domain Variant-Fc DomainVariant Fusion Polypeptides SEQ ID NO: Amino Acid Sequence 138EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 139DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 140EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 141DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 142EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 143EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 144QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCRKTHTCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 145EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSEKTHTCPECPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 146EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 147DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVEINAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 148EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 149DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVEINAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In addition to the knob-into-hole strategy, in some embodiments,electrostatic steering is also used to control the dimerization of Fcdomains. Electrostatic steering refers to the utilization of favorableelectrostatic interactions between oppositely charged amino acids inpeptides, protein domains, and proteins to control the formation ofhigher ordered protein molecules. In particular, to control thedimerization of Fc domains using electrostatic steering, one or moreamino acid residues that make up the CH3-CH3 interface are replaced withpositively- or negatively-charged amino acid residues such that theinteraction becomes electrostatically favorable or unfavorable dependingon the specific charged amino acids introduced. In some embodiments, apositively-charged amino acid in the interface, such as lysine,arginine, or histidine, is replaced with a negatively-charged amino acidsuch as aspartic acid or glutamic acid. In some embodiments, anegatively-charged amino acid in the interface is replaced with apositively-charged amino acid. In some embodiments, the charged aminoacids are introduced to one of the interacting CH3 antibody constantdomains, or both. In some embodiments, introducing charged amino acidsto the interacting CH3 antibody constant domains of the two Fc domainspromotes the selective formation of heterodimers of Fc domains ascontrolled by the electrostatic steering effects resulting from theinteraction between charged amino acids. Examples of electrostaticsteering amino acid pairs are included, without limitation, in Table 11.

TABLE 11 Electrostatic Steering Amino Acid Pairs Fc K409D K409D K409EK409E K392D K392D K392E K392E K409D K370E domain K392D K409D monomer 1K439E Fc D399K D399R D399K D399R D399K D399R D399K D399R D399K D356Kdomain D356K E357K monomer 2 D399K

Other methods used to control the heterodimerization of Fc domains,especially in the context of constructing a bispecific antibody, areavailable.

In some embodiments, a first Fc domain and a second Fc domain eachincludes one or more of the following amino acid substitutions: T366W,T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T,L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R,E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E,K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N,F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, andK409I, relative to the sequence of human IgG1.

In some embodiments an Fc domain comprises: (a) one of the followingamino acid substitutions relative to wild type human IgG1: T366W, T366S,L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H,L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q,S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D,T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T,F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I; or (b)(i) a N297A mutation relative to a human IgG1 Fc region; (ii) a L234A,L235A, and G237A mutation relative to a human IgG1 Fc region; (iii) aL234A, L235A, G237A, and N297A mutation relative to a human IgG1 Fcregion; (iv) a N297A mutation relative to a human IgG2 Fc region; (v) aA330S and P331S mutation relative to a human IgG2 Fc region; (vi) aA330S, P331S, and N297A mutation relative to a human IgG2 Fc region;(vii) a S228P, E233P, F234V, L235A, and deG236 mutation relative to ahuman IgG4 Fc region; or (viii) a S228P, E233P, F234V, L235A, deG236,and N297A mutation relative to a human IgG4 Fc region. In someembodiments an Fc domain variant comprises: (a) one of the followingamino acid substitutions relative to wild type human IgG1: T366W, T366S,L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H,L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q,S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D,T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T,F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I; and(b) further comprises (i) a N297A mutation relative to a human IgG1 Fcregion; (ii) a L234A, L235A, and G237A mutation relative to a human IgG1Fc region; (iii) a L234A, L235A, G237A, and N297A mutation relative to ahuman IgG1 Fc region; (iv) a N297A mutation relative to a human IgG2 Fcregion; (v) a A330S and P331S mutation relative to a human IgG2 Fcregion; (vi) a A330S, P331S, and N297A mutation relative to a human IgG2Fc region; (vii) a S228P, E233P, F234V, L235A, and delG236 mutationrelative to a human IgG4 Fc region; or (viii) a S228P, E233P, F234V,L235A, delG236, and N297A mutation relative to a human IgG4 Fc region.

In some embodiments, the first and second Fe domains include differentamino acid substitutions. In some embodiments, the first Fe domainincludes T366W. In some embodiments, the second Fe domain includesT366S, L368A, and Y407V. In some embodiments, the first Fe domainincludes D399K. In some embodiments, the second Fe domain includesK409D.

Linkers

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRPα D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRPα D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRPα D1 domain at a residueselected from the group consisting of residue 6, residue 27, residue 31,residue 47, residue 53, residue 54, residue 56, residue 66, and residue92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fe variant, wherein the Fe variant comprises an Fe domain dimercomprising two Fe domain variants, wherein each Fe domain variantindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

In the present disclosure, a linker is used to describe a linkage orconnection between polypeptides or protein domains or associatednon-protein moieties. In some embodiments, a linker is a linkage orconnection between an Fe domain (or variant thereof) and a SIRPα D1domain variant. In some embodiments, the linker connects the C-terminusof the SIRPα D1 domain variant and the N-terminus of the Fe domainvariant, such that the two polypeptides are joined to each other intandem series.

In some embodiments, a linker is a simple covalent bond, e.g., a peptidebond, a synthetic polymer, or any kind of bond created from a chemicalreaction, e.g. chemical conjugation. When a linker is a peptide bond, insome embodiments, the carboxylic acid group at the C-terminus of oneprotein domain reacts with the amino group at the N-terminus of anotherprotein domain in a condensation reaction to form a peptide bond. Insome embodiments, the peptide bond is formed from synthetic meansthrough a conventional organic chemistry reaction, or by naturalproduction from a host cell, wherein a nucleic acid molecule encodingthe DNA sequences of both proteins (e.g., an Fc domain variant and aSIRPα D1 domain variant) in tandem series can be directly transcribedand translated into a contiguous polypeptide encoding both proteins bythe necessary molecular machineries (e.g., DNA polymerase and ribosome)in the host cell.

When a linker is a synthetic polymer, in some embodiments, the polymeris functionalized with reactive chemical functional groups at each endto react with the terminal amino acids at the connecting ends of twoproteins.

When a linker (except peptide bond mentioned above) is made from achemical reaction, in some embodiments, chemical functional groups(e.g., amine, carboxylic acid, ester, azide, or other functionalgroups), are attached synthetically to the C-terminus of one protein andthe N-terminus of another protein, respectively. In some embodiments,the two functional groups then react through synthetic chemistry meansto form a chemical bond, thus connecting the two proteins together.

Spacers

In the present disclosure, in some embodiments, a linker between an Fcdomain monomer and a SIRPα D1 variant polypeptide of the disclosure, isan amino acid spacer including about 1-200 amino acids. Suitable peptidespacers include peptide linkers containing flexible amino acid residuessuch as glycine and serine. Examples of linker sequences are provided inTable 12. In some embodiments, a spacer contains motifs, e.g., multipleor repeating motifs, of GS, GG, GGS, GGG, GGGGS (SEQ ID NO: 163), GGSG(SEQ ID NO: 164), or SGGG (SEQ ID NO: 165). In some embodiments, aspacer contains 2 to 12 amino acids including motifs of GS, e.g., GS,GSGS (SEQ ID NO: 166), GSGSGS (SEQ ID NO: 167), GSGSGSGS (SEQ ID NO:168), GSGSGSGSGS (SEQ ID NO: 169), or GSGSGSGSGSGS (SEQ ID NO: 170). Insome embodiments, a spacer contains 3 to 12 amino acids including motifsof GGS, e.g., GGS, GGSGGS (SEQ ID NO: 171), GGSGGSGGS (SEQ ID NO: 172),and GGSGGSGGSGGS (SEQ ID NO: 173). In some embodiments, a spacercontains 4 to 12 amino acids including motifs of GGSG (SEQ ID NO: 164),e.g., GGSG (SEQ ID NO: 164), GGSGGGSG (SEQ ID NO: 174), or GGSGGGSGGGSG(SEQ ID NO: 175). In some embodiments, a spacer contains motifs of GGGGS(SEQ ID NO: 163), e.g., GGGGSGGGGSGGGGS (SEQ ID NO: 176). In someembodiments, a spacer contains amino acids other than glycine andserine, e.g., AAS (SEQ ID NO: 177), AAAL (SEQ ID NO: 178), AAAK (SEQ IDNO: 179), AAAR (SEQ ID NO: 180), EGKSSGSGSESKST (SEQ ID NO: 181),GSAGSAAGSGEF (SEQ ID NO: 182), AEAAAKEAAAKA (SEQ ID NO: 183),KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), GGGGAGGGG (SEQ ID NO: 185),GENLYFQSGG (SEQ ID NO: 186), SACYCELS (SEQ ID NO: 187), RSIAT (SEQ IDNO: 188), RPACKIPNDLKQKVMNH (SEQ ID NO: 189),GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 190), AAANSSIDLISVPVDSR(SEQ ID NO: 191), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO:192).

In some embodiments, a spacer contains motifs, e.g., multiple orrepeating motifs, of EAAAK (SEQ ID NO: 193). In some embodiments, aspacer contains motifs, e.g., multiple or repeating motifs, ofproline-rich sequences such as (XP)n, in which X is any amino acid(e.g., A, K, or E) and n is from 1-5, and PAPAP(SEQ ID NO: 194).

TABLE 12 Linker Sequences SEQ ID NO: AminoAcidSequence 163 GGGGS 164GGSG 165 SGGG 166 GSGS 167 GSGSGS 168 GSGSGSGS 169 GSGSGSGSGS 170GSGSGSGSGSGS 171 GGSGGS 172 GGSGGSGGS 173 GGSGGSGGSGGS 174 GGSGGGSG 175GGSGGGSGGGSG 176 GGGGSGGGGSGGGGS 177 AAS 178 AAAL 179 AAAK 180 AAAR 181EGKSSGSGSESKST 182 GSAGSAAGSGEF 183 AEAAAKEAAAKA 184 KESGSVSSEQLAQFRSLD185 GGGGAGGGG 186 GENLYFQSGG 187 SACYCELS 188 RSIAT 189RPACKIPNDLKQKVMNH 190 GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG 191AAANSSIDLISVPVDSR 192 GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS 193 EAAAK 194PAPAP

In some embodiments, the length of the peptide spacer and the aminoacids used is adjusted depending on the two proteins involved and thedegree of flexibility desired in the final protein fusion polypeptide.In some embodiments, the length of the spacer is adjusted to ensureproper protein folding and avoid aggregate formation. In someembodiments, a spacer is A or AAAL (SEQ ID NO: 178).

Vectors, Host Cells, and Protein Production

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SRP-α) D1 variant comprising a SIRPα D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRPα D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRPα D1 domain at a residueselected from the group consisting of residue 6, residue 27, residue 31,residue 47, residue 53, residue 54, residue 56, residue 66, and residue92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

In some embodiments, the polypeptides of the disclosure are producedfrom a host cell. A host cell refers to a vehicle that includes thenecessary cellular components, e.g., organelles, needed to express thepolypeptides and fusion polypeptides described herein from theircorresponding nucleic acids. In some embodiments, the nucleic acids areincluded in nucleic acid vectors introduced into the host cell bytransformation, transfection, electroporation, calcium phosphateprecipitation, direct microinjection, infection, etc. In someembodiments, the choice of nucleic acid vector depends on the host cellto be used. In some embodiments, host cells are of either prokaryotic(e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

In some embodiments, a polypeptide, for example a polypeptide constructcomprising a SIRPα D1 domain variant (e.g., any variant provided inTables 2, 5, and 6) and a fusion partner such as an Fc variant areproduced by culturing a host cell transformed with a nucleic acid,preferably an expression vector, containing a nucleic acid encoding thepolypeptide construct (e.g., Fc variant, linker, and fusion partner)under the appropriate conditions to induce or cause expression of thepolypeptide construct. In some embodiments, the conditions appropriatefor expression varies with the expression vector and the host cellchosen. In some embodiments, a wide variety of appropriate host cellsare used, including, but not limited to, mammalian cells, bacteria,insect cells, and yeast. For example, a variety of cell lines that finduse in the present disclosure are described in the ATCC® cell linecatalog, available from the American Type Culture Collection. In someembodiments, Fc domain variants of this disclosure are expressed in acell that is optimized not to glycosylate proteins that are expressed bysuch cell, either by genetic engineering of the cell line ormodifications of cell culture conditions such as addition of kifunensineor by using a naturally non-glycosylating host such as a prokaryote (E.coli, etc.), and in some cases, modification of the glycosylationsequence in the Fc is not be needed.

Nucleic Acid Vector Construction and Host Cells

A nucleic acid sequence encoding the amino acid sequence of apolypeptide of the disclosure can be prepared by a variety of methods.These methods include, but are not limited to, oligonucleotide-mediated(or site-directed) mutagenesis and PCR mutagenesis. In some embodiments,a nucleic acid molecule encoding a polypeptide of the disclosure isobtained using standard techniques, e.g., gene synthesis. Alternatively,a nucleic acid molecule encoding a wild-type SIRPα D1 domain is mutatedto include specific amino acid substitutions using standard techniques,e.g., QuikChange™ mutagenesis. In some cases, nucleic acid molecules aresynthesized using a nucleotide synthesizer or PCR techniques.

In some embodiments, the nucleic acids that encode a polypeptideconstruct, for example a polypeptide construct comprising a SIRPα D1domain variant (e.g., any variant provided in Tables 2, 5, and 6) and afusion partner such as an Fc variant are incorporated into an expressionvector in order to express the protein. A variety of expression vectorscan be utilized for protein expression. Expression vectors can compriseself-replicating, extra-chromosomal vectors or vectors which integrateinto a host genome. A vector can also include various components orelements. For example, in some embodiments, the vector componentsinclude, but are not limited to, transcriptional and translationalregulatory sequences such as a promoter sequence, a ribosomal bindingsite, a signal sequence, transcriptional start and stop sequences,translational start and stop sequences, 3′ and 5′ untranslated regions(UTRs), and enhancer or activator sequences; an origin of replication; aselection marker gene; and the nucleic acid sequence encoding thepolypeptide of interest, and a transcription termination sequence. Insome embodiments, expression vectors comprise a protein operably linkedwith control or regulatory sequences, selectable markers, any fusionpartners, additional elements, or any combinations thereof. The term“operably linked” means that the nucleic acid is placed into afunctional relationship with another nucleic acid sequence. Generally,these expression vectors include transcriptional and translationalregulatory nucleic acid operably linked to the nucleic acid encoding theFc variant, and are typically appropriate to the host cell used toexpress the protein. A selection gene or marker, such as, but notlimited to, an antibiotic resistance gene or fluorescent protein gene,can be used to select for host cells containing the expression vector,for example by antibiotic or fluorescence expression. Various selectiongenes are available.

In some embodiments, the components or elements of a vector areoptimized such that expression vectors are compatible with the host celltype. Expression vectors which find use in the present disclosureinclude, but are not limited to, those which enable protein expressionin mammalian cells, bacteria, insect cells, yeast, and in in vitrosystems.

In some embodiments, mammalian cells are used as host cells to producepolypeptides of the disclosure. Examples of mammalian cell typesinclude, but are not limited to, human embryonic kidney (HEK) (e.g.,HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero,MC3T3, NSO, Sp2/0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20,T47D, NSO (a murine myeloma cell line that does not endogenously produceany immunoglobulin chains), CRL7030, and HsS78Bst cells. In someembodiments, E. coli cells are used as host cells to producepolypeptides of the disclosure. Examples of E. coli strains include, butare not limited to, E. coli 294 (ATCC®31,446), E. coli λ 1776 (ATCC®31,537, E. coli BL21 (DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC®31,608).

Different host cells have characteristic and specific mechanisms for theposttranslational processing and modification of protein products (e.g.,glycosylation). In some embodiments, appropriate cell lines or hostsystems are chosen to ensure the correct modification and processing ofthe polypeptide expressed. Once the vectors are introduced into hostcells for protein production, host cells are cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

In some embodiments, a polypeptide construct, for example a polypeptideconstruct comprising a SIRPα D1 domain variant (e.g., any variantprovided in Tables 2, 5, and 6) and a fusion partner such as an Fcvariant are expressed in mammalian expression systems, including systemsin which the expression constructs are introduced into the mammaliancells using virus such as retrovirus or adenovirus. In some embodiments,human, mouse, rat, hamster, or primate cells are utilized. Suitablecells also include known research cells, including but not limited toJurkat T cells, NIH3T3, CHO, COS, and 293 cells. Alternately, in someembodiments, proteins are expressed in bacterial cells. Bacterialexpression systems are well known in the art, and include Escherichiacoli (E. coli), Bacillus subtilis, Streptococcus cremoris, andStreptococcus lividans. In some cases, polypeptide constructs comprisingFc domain variants are produced in insect cells such as but not limitedto Sf9 and Sf21 cells or yeast cells such as but not limited toorganisms from the genera Saccharomyces, Pichia, Kluyveromyces,Hansenula and Yarrowia. In some cases, polypeptide constructs comprisingFc domain variants are expressed in vitro using cell free translationsystems. In vitro translation systems derived from both prokaryotic(e.g., E. coli) and eukaryotic (e.g., wheat germ, rabbit reticulocytes)cells are available and, in some embodiments, chosen based on theexpression levels and functional properties of the protein of interest.For example, as appreciated by those skilled in the art, in vitrotranslation is required for some display technologies, for exampleribosome display. In addition, in some embodiments, the Fc domainvariants are produced by chemical synthesis methods such as, but notlimited to, liquid-phase peptide synthesis and solid-phase peptidesynthesis. In the case of in vitro transcription using anon-glycosylating system such as bacterial extracts, the Fc will not beglycosylated even in presence of the natural glycosylation site andtherefore inactivation of the Fc will be equivalently obtained.

In some embodiments, a polypeptide construct includes non-natural aminoacids, amino acid analogues, amino acid mimetics, or any combinationsthereof that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids generally refer to the 20common amino acids (alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine.Amino acid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, e.g., a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,such as, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. In some embodiments, such analogs have modified R groups(such as, norleucine) or modified peptide backbones, but generallyretain the same basic chemical structure as a naturally occurring aminoacid.

Protein Production, Recovery, and Purification

In some embodiments, host cells used to produce polypeptides of thedisclosure are grown in media suitable for culturing of the selectedhost cells. Examples of suitable media for mammalian host cells includeMinimal Essential Medium (MEM), Dulbecco's Modified Eagle's Medium(DMEM), Expi293™ Expression Medium, DMEM with supplemented fetal bovineserum (FBS), and RPMI-1640. Examples of suitable media for bacterialhost cells include Luria broth (LB) plus necessary supplements, such asa selection agent, e.g., ampicillin. In some embodiments, host cells arecultured at suitable temperatures, such as from about 20° C. to about39° C., e.g., from about 25° C. to about 37° C., preferably 37° C., andCO₂ levels, such as about 5% to 10%. In some embodiments, the pH of themedium is from about pH 6.8 to pH 7.4, e.g., pH 7.0, depending mainly onthe host organism. If an inducible promoter is used in the expressionvector, protein expression can be induced under conditions suitable forthe activation of the promoter.

In some embodiments, protein recovery involves disrupting the host cell,for example by osmotic shock, sonication, or lysis. Once the cells aredisrupted, cell debris is removed by centrifugation or filtration. Theproteins can then be further purified. In some embodiments, apolypeptide of the disclosure is purified by various methods of proteinpurification, for example, by chromatography (e.g., ion exchangechromatography, affinity chromatography, and size-exclusion columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. For example,in some embodiments, the protein is isolated and purified byappropriately selecting and combining affinity columns such as Protein Acolumn (e.g., POROS Protein A chromatography) with chromatographycolumns (e.g., POROS HS-50 cation exchange chromatography), filtration,ultra-filtration, de-salting and dialysis procedures. In someembodiments, a polypeptide is conjugated to marker sequences, such as apeptide to facilitate purification. An example of a marker amino acidsequence is a hexa-histidine peptide (His6-tag (SEQ ID NO: 223)), whichcan bind to a nickel-functionalized agarose affinity column withmicromolar affinity. As an alternative, a hemagglutinin “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein can be used.

In some embodiments, polypeptides of the disclosure, for example apolypeptide construct comprising a SIRPα D1 domain variant (e.g., anyvariant provided in Tables 2, 5, and 6) and a fusion partner such as anFc variant are produced by the cells of a subject (e.g., a human), e.g.,in the context of gene therapy, by administrating a vector such as aviral vector (e.g., a retroviral vector, adenoviral vector, poxviralvector (e.g., vaccinia viral vector, such as Modified Vaccinia Ankara(MVA)), adeno-associated viral vector, and alphaviral vector) containinga nucleic acid molecule encoding a polypeptide of the disclosure. Thevector, once inside a cell of the subject (e.g., by transformation,transfection, electroporation, calcium phosphate precipitation, directmicroinjection, infection, etc.) can be used for the expression of apolypeptide disclosed herein. In some cases, the polypeptide is secretedfrom the cell. In some embodiments, if treatment of a disease ordisorder is the desired outcome, no further action is required. In someembodiments, if collection of the protein is desired, blood is collectedfrom the subject and the protein purified from the blood by variousmethods.

Methods of Treating Cancer

Provided herein is a method of treating cancer in an individual (e.g., ahuman individual) that comprises administering to the individual aneffective amount of (a) a polypeptide comprising a SIRPα D1 domainvariant (e.g., a SIRPα D1 domain variant described herein) and an Fcdomain variant (e.g., an Fc domain variant described herein) and (b) atherapeutic antibody.

Lung Cancer

In some embodiments, provided is a method of treating lung cancer (e.g.,non-small cell lung cancer or “NSCLC”), in an individual (e.g., a humanindividual) that comprises administering to the individual an effectiveamount of (a) a polypeptide (e.g., fusion polypeptide) comprising aSIRPα D1 domain variant (e.g., a SIRPα D1 domain variant describedherein) and an Fc domain variant (e.g., an Fc domain variant describedherein) and (b) a therapeutic antibody that disrupts the interactionbetween PD-1 and PD-L1, wherein the individual progressed (e.g.,demonstrated disease progression) while on (or following) a priortherapy for lung cancer (e.g., NSCLC). In some embodiments, the priortherapy was immune checkpoint inhibitor (CPI) therapy. Additionally oralternatively, in some embodiments, the individual has a PD-L1 tumorproportion score (TPS) of less than 50%. In some embodiments, theindividual has not received prior CPI therapy. In some embodiments, thepolypeptide (e.g., fusion polypeptide) comprises a SIRPα D1 domainvariant that comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgGFc region comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (ii) a human IgG2 Fcregion comprising A330S, P331S, and N297A mutations, wherein numberingis according to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and deG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments the polypeptide (e.g., fusion polypeptide) administeredto the individual comprises a SIRPα D1 domain variant that comprises theamino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In someembodiments the polypeptide (e.g., fusion polypeptide) administered tothe individual comprises an Fc domain variant that is a human IgG Fcregion comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat. In some embodiments,the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises the amino acid sequence of SEQID NO: 136 or SEQ ID NO: 135. In some embodiments the polypeptide (e.g.,fusion polypeptide) forms a homodimer. In some embodiments, thetherapeutic antibody that blocks the interaction between PD-1 and PD-L1is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody ispembrolizumab.

In some embodiments, the pembrolizumab is administered subcutaneously.In some embodiments, the pembrolizumab is administered via intravenousinfusion. In some embodiments, the pembrolizumab is administeredaccording to its label instructions. In some embodiments, thepembrolizumab is administered to the individual (e.g., via IV infusion)at a dose of about 200 mg every three weeks (Q3W). In some embodiments,the pembrolizumab is administered to the individual for up to 24 months.In some embodiments, the pembrolizumab is administered to the individualfor at least 24 months. In some embodiments, dose modifications ofpembrolizumab are made according to the local package insert. Completeinformation about pembrolizumab preparation, dispensing, dosage, andadministration schedule can be found in the local package insert (forthe United States, see, e.g.,www(dot)accessdata(dot)fda(dot)gov/drugsatfda_docs/label/2016/125514s0121bl(dot)pdf,for Europe, see, e.g.,www(dot)ema(dot)europa(dot)eu/en/documents/product-information/keytruda-epar-product-information_en(dot)pdf).In some embodiments, the polypeptide (e.g., fusion polypeptide) isadministered (e.g., via intravenous infusion) to the individual weekly(i.e., once every 7 days or “qw”), e.g., at a dose of 10.0 mg/kg.

In some embodiments, the lung cancer is NSCLC. In some embodiments, theNSCLC is locally advanced NSCLC. In some embodiments, the NSCLC ismetastatic NSCLC. In some embodiments, the individual has metastaticNSCLC and has not demonstrated disease progression within 8 weeks of thestart of a prior therapy with a PD-1 or PD-L1 inhibitor. In someembodiments, the individual has a PD-L1 tumor proportion score (TPS)score of ≥1%. In some embodiments, the individual has locally advancedor metastatic NSCLC with a TPS score <50%. In some embodiments, theindividual progressed following systemic therapy for their metastaticdisease. In some embodiments, the individual has locally advanced ormetastatic NSCLC with a TPS score ≥1%, and the individual progressed onprior checkpoint inhibitor (CPI) therapy for NSCLC.

In some embodiments, the prior CPI therapy for lung cancer (e.g., NSCLC)on which the individual progressed was a therapy that comprisedtreatment with nivolumab, pembrolizumab, atezolizumab, avelumab,durvalumab, cemiplimab, tislelizumab (also known as BGB-A317),toripalimab, sintilimab, camrelizumab (also known as SHR-1210 orINCSHR-1210), spartalizumab (also known as PDR001), TSR-042, and/orFAZ053. In some embodiments, the individual is considered to haveprogressed on the prior CPI therapy for lung cancer (e.g., NSCLC) if theindividual demonstrated progressive disease (PD), e.g., as assessed byResponse Evaluation In Solid Tumor (RECIST) criteria (e.g., version 1.0or 1.1) or modified RECIST criteria (see, e.g., Therasse et al. (2000) JNatl Cancer Inst. 92: 205-216; Eisenhauer et al. (2009) Eur J Cancer.45: 229-247; and Jang et al. (2013) Chin J Cancer Res. 25(6): 689-694),World Health Organization (WHO) criteria (see, e.g., WHO. Handbook forReporting Results of Cancer Treatment. Geneva: World Health OrganizationOffset Publication; 1979. p. 48; and Miller et al. (1981) Cancer. 47:207-214), or any set of response criteria described in Hwang et al.(2017) “Response Evaluation of Chemotherapy for Lung Cancer.” TubercRespir Dis (Seoul). 80(2): 136-142 and references cited therein. In someembodiments, the individual is resistant to standard therapy (e.g.,curative therapy) for lung cancer (e.g., NSCLC). In some embodiments,there is no standard therapy (e.g., curative therapy) available to treatthe lung cancer (e.g., NSCLC).

In some embodiments, the individual's PD-L1 tumor proportion score (TPS)is assessed using an in vitro diagnostic immunohistochemistry (IHC)assay for detection of PD-L1 in formalin-fixed, paraffin-embedded (FFPE)human tissue sections. In some embodiments, the IHC assay is PD-L1 IHC22C3 Pharm Dx. PD-L1 IHC 22C3 Pharm Dx is a qualitativeimmunohistochemical assay in which a murine monoclonal anti-PD-L1antibody (clone 22C3) is used to detect PD-L1 (i.e., human PD-L1) informalin-fixed, paraffin-embedded (FFPE) lung cancer tissue (e.g., NSCLCtissue) obtained from an individual (e.g., patient) on the DAKO™AUTOSTAINER LINK 48 automated staining system using the ENVISION™ FLEXvisualization system. In some embodiments, TPS is a measure of PD-L1protein expression in the lung cancer (e.g., NSCLC) tissue sample fromthe individual (e.g., patient). In some embodiments, TPS is thepercentage of viable tumor cells showing partial or complete membranestaining at any intensity. The specimen should be considered PD-L1positive if TPS ≥50% of the viable tumor cells exhibit membrane stainingat any intensity. In some embodiments, tumor-associated immune cells(such as infiltrating lymphocytes or macrophages) are not included inthe scoring for the determination of TPS. In some embodiments thelabeling of the lung cancer (e.g., NSCLC) sample is performed by apathologist. In some embodiments, the staining is assessed via lightmicroscope at 10×-40× magnification. Further details regarding the PD-L1IHC 22C3 Pharm Dx assay, reagents and equipment to perform the assay,interpretation of assay results, and TPS scoring are provided atwww(dot)accessdata(dot)fda(dot)gov/cdrh_docs/pdf15/p150013b(dot)pdf andReck et al. (2016) “Pembrolizurnab versus Chemotherapy forPD-L1-Positive Non-Small-Cell Lung Cancer.” NEJM. 375: 1823-1833.

Head and Neck Cancer

In some embodiments, provided is a method of treating head and neckcancer (e.g., head and neck squamous cell carcinoma or “HNSSC”) in anindividual (e.g., a human individual) that comprises administering tothe individual an effective amount of (a) a polypeptide (e.g., fusionpolypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1domain variant described herein) and an Fc domain variant (e.g., an Fcdomain variant described herein) and (b) a therapeutic antibody thatdisrupts the interaction between PD-1 and PD-L1, wherein the individualprogressed (e.g., demonstrated disease progression) while on a priortherapy or following a prior therapy for head and neck cancer (e.g.,HNSCC). In some embodiments, the prior therapy was a platinum-containingtherapy. In some embodiments, the polypeptide (e.g., fusion polypeptide)comprises a SIRPα D1 domain variant that comprises the amino acidsequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fcdomain variant is (i) a human IgG Fc region comprising L234A, L235A,G237A, and N297A mutations, wherein numbering is according to the EUindex of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, andN297A mutations, wherein numbering is according to the EU index ofKabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, and delG236 mutations, wherein numbering is according to the EUindex of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P,F234V, L235A, deG236, and N297A mutations, wherein numbering isaccording to the EU index of Kabat. In some embodiments the polypeptide(e.g., fusion polypeptide) administered to the individual comprises aSIRPα D1 domain variant that comprises the amino acid sequence of SEQ IDNO: 81 or SEQ ID NO: 85. In some embodiments the polypeptide (e.g.,fusion polypeptide) administered to the individual comprises an Fedomain variant that is a human IgG Fc region comprising L234A, L235A,G237A, and N297A mutations, wherein numbering is according to the EUindex of Kabat. In some embodiments, the Fc domain variant comprises theamino acid sequence of SEQ ID NO: 91. In some embodiments, thepolypeptide (e.g., fusion polypeptide) administered to the individualcomprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135.In some embodiments the polypeptide (e.g., fusion polypeptide) forms ahomodimer. In some embodiments, the therapeutic antibody that blocks theinteraction between PD-1 and PD-L1 is an anti-PD-1 antibody. In someembodiments, the anti-PD-1 antibody is pembrolizumab. In someembodiments, the HNSCC is PD-L1 negative. In some embodiments, the HNSCCis PD-L1 positive. Further details regarding “PD-L1 negative” and “PD-L1positive” are provided elsewhere herein

In some embodiments, the pembrolizumab is administered subcutaneously.In some embodiments, the pembrolizumab is administered via intravenousinfusion. In some embodiments, the pembrolizumab is administeredaccording to its label instructions. In some embodiments, thepembrolizumab is administered to the individual (e.g., via IV infusion)at a dose of about 200 mg every three weeks (Q3W). In some embodiments,the pembrolizumab is administered to the individual for up to 24 months.In some embodiments, the pembrolizumab is administered to the individualfor at least 24 months. In some embodiments, dose modifications ofpembrolizumab are made according to the local package insert. Completeinformation about pembrolizumab preparation, dispensing, dosage, andadministration schedule can be found in the local package insert (forthe United States, see, e.g.,www(dot)accessdata(dot)fda(dot)gov/drugsatfda_docs/label/2016/125514s021bl(dot)pdf,for Europe, see, e.g.,www(dot)ema(dot)europa(dot)eu/en/documents/product-information/keytruda-epar-product-information_en(dot)pdf).In some embodiments, the polypeptide (e.g., fusion polypeptide) isadministered (e.g., via intravenous infusion) to the individual weekly(i.e., once every 7 days or “qw”), e.g., at a dose of 10.0 mg/kg.

In some embodiments, the individual has recurrent HNSCC. In someembodiments, the HNSCC is metastatic HNSCC. In some embodiments, theindividual received prior therapy with an immune checkpoint inhibitor(“CPI”), i.e., the individual is a “CPI experienced” individual. In someembodiments, the individual has recurrent or metastatic HNSCC and hasnot demonstrated with disease progression within 8 weeks of the start ofa prior therapy with a CPI. In some embodiments, the prior CPI therapywas or comprised a PD-1 or PD-L1 inhibitor. In some embodiments, theprior CPI was or comprised treatment with nivolumab, pembrolizumab,atezolizumab, avelumab, durvalumab, cemiplimab, tislelizumab (also knownas BGB-A317), toripalimab, sintilimab, camrelizumab (also known asSHR-1210 or INCSHR-1210), spartalizumab (also known as PDR001), TSR-042,and/or FAZ053. In some embodiments, the individual has not receivedprior therapy with an CPI (e.g., the individual is “immune checkpointinhibitor naïve” or “CPI naïve”).

In some embodiments, the prior platinum-containing therapy on which theindividual progressed or following which the individual progressed was atherapy that comprised treatment with carboplatin, cisplatin,oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin,picoplatin, and/or satraplatin. In some embodiments, the individual isconsidered to have progressed on or following the priorplatinum-containing therapy for head and neck cancer (e.g., HNSCC) ifthe individual demonstrated progressive disease (PD), e.g., as assessedby Response Evaluation In Solid Tumor (RECIST) criteria (e.g., version1.0 or 1.1) or modified RECIST criteria (see, e.g., Therasse et al.(2000) J Natl Cancer Inst. 92: 205-216; Eisenhauer et al. (2009) Eur JCancer. 45: 229-247; and Jang et al. (2013) Chin J Cancer Res. 25(6):689-694), World Health Organization (WHO) criteria (see, e.g., WHO.Handbook for Reporting Results of Cancer Treatment. Geneva: World HealthOrganization Offset Publication; 1979. p. 48; and Miller et al. (1981)Cancer. 47: 207-214), or a set of response criteria described in Wray etal. (2016) “Therapy Response Assessment and Patient Outcomes in Head andNeck Squamous Cell Carcinoma: FDG PET Hopkins Criteria Versus ResidualNeck Node Size and Morphologic Features.” Am J Roentgenology.207:641-647. In some embodiments, the individual is resistant tostandard therapy (e.g., curative therapy) for head and neck cancer(e.g., HNSCC). In some embodiments, there is no standard therapy (e.g.,curative therapy) available to treat the head and neck cancer (e.g.,HNSCC).

Gastric/Gastroesophageal (GEJ) Cancer

In some embodiments, provided is a method of treatinggastric/gastroesophageal (GEJ) cancer (e.g., HER2-positive gastric orGEJ adenocarcinoma) in an individual (e.g., a human individual) thatcomprises administering to the individual an effective amount of (a) apolypeptide (e.g., fusion polypeptide) comprising a SIRPα D1 domainvariant (e.g., a SIRPα D1 domain variant described herein) and an Fcdomain variant (e.g., an Fc domain variant described herein) and (b) atherapeutic anti-HER2 antibody, wherein the individual progressed (e.g.,demonstrated disease progression) during a prior therapy or following aprior therapy for gastric/GEJ cancer (e.g., gastric/GEJ adenocarcinoma),and wherein the prior therapy was an anti-HER2 antibody therapy and/or afluoropyrimidine-based therapy. In some embodiments, the polypeptide(e.g., fusion polypeptide) comprises a SIRPα D1 domain variant thatcomprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. Insome embodiments, the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises a SIRPα D1 domain variant thatcomprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. Insome embodiments the polypeptide (e.g., fusion polypeptide) administeredto the individual comprises an Fc domain variant that is a human IgG1 Fcregion comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat. In some embodiments,the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises the amino acid sequence of SEQID NO: 136 or SEQ ID NO: 135. In some embodiments the polypeptide (e.g.,fusion polypeptide) forms a homodimer. In some embodiments, thetherapeutic anti-HER2 antibody that administered to the individual incombination with the fusion polypeptide is trastuzumab.

In some embodiments, the trastuzumab is administered subcutaneously. Insome embodiments, the trastuzumab is administered via intravenousinfusion. In some embodiments, the trastuzumab is administered accordingto its label instructions. In some embodiments, the trastuzumab isadministered to the individual (e.g., via intravenous infusion) everythree weeks (Q3W). In some embodiments, the initial (i.e., first) doseof trastuzumab is about 8 mg/kg, and every subsequent dose (i.e.,following the first dose) is about 6 mg/kg. In some embodiments, dosemodifications of trastuzumab are made according to the local packageinsert. Complete information about trastuzumab preparation, dispensing,dosage, and administration schedule can be found in the local packageinsert (for the United States, see, e.g.,www.accessdata(dot)fda(dot)gov/drugsatfda_docs/label/2017/103792s53371bl(dot)pdf; for Europe, see, e.g.,www(dot)ema(dot)Europa(dot)eu/en/documents/product-information/herceptin-epar-product-information_en(dot)pdf).In some embodiments, the polypeptide (e.g., fusion polypeptide) isadministered (e.g., via intravenous infusion) to the individual weekly(i.e., once every 7 days or “qw”), e.g., at a dose of 10.0 mg/kg.

In some embodiments HER2 status (i.e., HER2-positive status orHER2-negative status) of the gastric or GEJ cancer is assessed viaimmunohistochemistry (IHC) or in situ hybridization (ISH, e.g.,fluorescent ISH or “FISH”). In some embodiments, the HER2 status of thegastric or GEJ cancer is evaluated according to criteria described inAbrahao-Machado et al. (2016) World J Gastroenterol. 22(19): 4619-4625and references cited therein. In some embodiments, the HER2-positivegastric or HER2-positive GEJ cancer is HER2-positivegastric/HER2-positive GEJ adenocarcinoma. In some embodiments, theHER2-positive gastric/HER2-positive GEJ cancer (e.g., adenocarcinoma) ismetastatic gastric/GEJ cancer (e.g., metastatic adenocarcinoma). In someembodiments, the individual has metastatic gastric/GEJ cancer (e.g.,adenocarcinoma) and has demonstrated a response of at least stabledisease (SD) (i.e., a response better than progressive disease (PD)) asthe best response to a prior therapy.

In some embodiments, the individual progressed (e.g., demonstrateddisease progression) during prior therapy or following prior therapywith an anti-HER2 antibody. In some embodiments, the prior anti-HER2antibody therapy comprised treatment with trastuzumab, pertuzumab,and/or margetuximab. Additionally or alternatively, in some embodiments,the individual progressed (e.g., demonstrated disease progression)during prior therapy or following prior therapy with afluoropyrimidine-based therapy. In some embodiments the priorfluoropyrimidine-based therapy comprised treatment with, e.g.,capecitabine, floxuridine, 4-fluorouracil, 5-fluorouracil, carmofur,doxifluridine, ftorafur (Tegafur), UFT (i.e., a 1:4 molar combination offtorafur with uracil), S-1 (a combination of ftorafur, gimeracil, andoteracil), and/or FOLFOX (a combination of folinic acid, 5-fluorouracil,and oxaliplatin). In some embodiments, the individual progressed whileon therapy or following therapy with an anti-HER2 antibody and afluoropyrimidine-based therapy. In some embodiments, the anti-HER2antibody and the fluoropyrimidine-based therapy were administered incombination (e.g., wherein both agents were part of a single treatmentregimen). In some embodiments the anti-HER2 antibody and thefluoropyrimidine-based therapy were each administered in separatetreatment regimens (e.g., in two separate prior therapies or twoseparate prior lines of therapy).

In some embodiments, the individual is considered to have progressed onthe prior anti-HER2 antibody therapy and/or the priorfluoropyrimidine-based therapy for gastric/GEJ cancer (e.g.,HER2-positive gastric/GEJ cancer) if the individual demonstratedprogressive disease (PD), e.g., as assessed by Response Evaluation InSolid Tumor (RECIST) criteria (e.g., version 1.0 or 1.1) or modifiedRECIST criteria (see, e.g., Therasse et al. (2000) J Nat Cancer Inst.92: 205-216; Eisenhauer et al. (2009) Eur J. Cancer. 45: 229-247; andJang et al. (2013) Chin J. Cancer Res. 25(6): 689-694), World HealthOrganization (WHO) criteria (see, e.g., WHO. Handbook for ReportingResults of Cancer Treatment. Geneva: World Health Organization OffsetPublication; 1979. p. 48; and Miller et al. (1981) Cancer. 47: 207-214),or any set of response criteria described in Kurokawa et al. (2013) AnnSurg Oncol. 20(9): 3009-3014; Yanagawa et al. (2012) J Nucl Med. 53(6):872-880; Lordick et al. (2016) Ann Oncol. 27(suppl 5): v50-v57; or Kimet al. (2015) Oncology. 88:69-75. In some embodiments, the individual isresistant to standard therapy (e.g., curative therapy) for gastric/GEJcancer (e.g., HER2-positive gastric/HER2-positive GEJ cancer). In someembodiments, there is no standard therapy (e.g., curative therapy)available to treat the gastric/GEJ cancer (e.g., HER2-positivegastric/HER2-positive GEJ cancer).

Lymphomas

(i) Aggressive Non-Hodgkin Lymphoma

In some embodiments, provided is a method of treating aggressivenon-Hodgkin lymphoma or “NHL” (e.g., diffuse large B-cell lymphoma(“DLBCL”, e.g., de novo DLBCL or transformed DLBCL or mantle celllymphoma (MCL)) in an individual (e.g., a human individual) thatcomprises administering to the individual an effective amount of (a) apolypeptide (e.g., fusion polypeptide) comprising a SIRPα D1 domainvariant (e.g., a SIRPα D1 domain variant described herein) and an Fcdomain variant (e.g., an Fc domain variant described herein) and (b) atherapeutic anti-CD20 antibody, wherein the aggressive NHL is relapsedand/or refractory aggressive NHL (e.g., wherein the individual hasrelapsed during or following a prior treatment for aggressive NHL and/orhas been refractory to a prior treatment for aggressive NHL) and whereinthere is no available therapy (e.g., curative therapy) for theaggressive NHL (e.g., DLBCL, such as de novo DLBCL, transformed DLBCL,or mantle cell lymphoma). In some embodiments, the polypeptide (e.g.,fusion polypeptide) comprises a SIRPα D1 domain variant that comprisesthe amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In someembodiments, the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises a SIRPα D1 domain variant thatcomprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. Insome embodiments the polypeptide (e.g., fusion polypeptide) administeredto the individual comprises an Fc domain variant that is a human IgG1 Fcregion comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat. In some embodiments,the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises the amino acid sequence of SEQID NO: 136 or SEQ ID NO: 135. In some embodiments the polypeptide (e.g.,fusion polypeptide) forms a homodimer. In some embodiments, thetherapeutic anti-CD20 antibody is rituximab. In some embodiments, theaggressive NHL is diffuse large B-cell lymphoma (DLBCL), e.g., de novoDLBCL or transformed DLBCL. In some embodiments, the aggressive NHL ismantle cell lymphoma (MCL).

In some embodiments, the rituximab is administered subcutaneously. Insome embodiments, the rituximab is administered via intravenousinfusion. In some embodiments, the rituximab is administered accordingto its label instructions. In some embodiments, the rituximab isadministered to the individual (e.g., via intravenous infusion) at adose of about 375 mg/m². In some embodiments, the first four doses (i.e.doses 1-4) of rituximab are administered to the individual (e.g., at adose of about 375 mg/m²) once a week (e.g., once every 7 days or “qw”)for the first four weeks (e.g., 28 days) of treatment, and the nexteight doses (i.e., doses 5-12) are administered to the individual onceevery four weeks (e.g., every 28 days or “q4w”). In some embodiments,the first four doses (i.e. doses 1-4) of rituximab are administered tothe individual (e.g., at a dose of about 375 mg/m²) once a week (e.g.,once every 7 days or “qw”) for the first four weeks (e.g., 28 days) oftreatment, and the next four doses (i.e., doses 5-8) are administered tothe individual once every four weeks (e.g., every 28 days or “q4w”). Insome embodiments, dose modifications of rituximab are made according tothe local package insert. Complete information about rituximabpreparation, dispensing, dosage, and administration schedule can befound in the local package insert (for the United States, see, e.g.,www(dot)accessdata(dot)fda(dot)gov/drugsatfda_docs/label/2012/103705s5367s53881bl(dot)pdf,for Europe, see, e.g.,www(dot)ema(dot)europa(dot)eu/en/documents/product-information/mabthera-epar-product-information_en(dot)pdf).In some embodiments, the polypeptide (e.g., fusion polypeptide) isadministered (e.g., via intravenous infusion) to the individual weekly(i.e., once every 7 days or “qw”), e.g., at a dose of 10.0 mg/kg or 15.0mg/kg.

In some embodiments, the individual is diagnosed as having de novo DLBCL(e.g., de novo relapsed and/or refractory DLBCL) if the individual hadno prior history of lymphoma. In some embodiments, the individual isdiagnosed as having transformed DLBCL if the individual has a history oflymphoma, e.g., indolent lymphoma, such as marginal zone lymphoma,lymphoplasmacytic lymphoma, small lymphocytic lymphoma/chroniclymphocytic leukemia, follicular lymphoma, or lymphocyte predominantHodgkin lymphoma. In some embodiments, the individual is diagnosed ashaving mantle cell lymphoma (MCL) if the individual is found to have oneor more of the following chromosomal abnormalities: t(11;14), t(14;18).In some embodiments, the individual is diagnosed with mantle celllymphoma (MCL) if SOX11 overexpression is detected in a sample ofleukemic cells from the individual. Other criteria for diagnosing denovo DLBCL, transformed DLBCL, and MCL are known in the art androutinely used by skilled artisans. See, e.g., Balsas et al. (2017)Blood. 130(4):501-513; National Guideline Alliance (UK). Non-Hodgkin'sLymphoma: Diagnosis and Management. London: National Institute forHealth and Care Excellence (UK); 2016 Jul. (NICE Guideline, No. 52.) 3,Staging; Dreyling et al. Am Soc Clin Oncol Educ Book. 2014:191-8; andothers.

In some embodiments, the individual is considered to have relapsedfollowing a prior therapy for aggressive NHL (e.g., de novo DLBCL,transformed DLBCL, or MCL) if the individual achieved a therapeuticresponse of least stable disease (SD) to the prior therapy, but stoppedresponding (e.g., demonstrated disease progression) within about any oneof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months following thecessation of the prior therapy. In some embodiments, the individual isconsidered to be refractory to a prior therapy for aggressive NHL if theindividual was unresponsive to the prior therapy (e.g., failed toachieve a therapeutic response of at least stable disease (SD) during orfollowing the prior therapy). In some embodiments, the therapeuticresponse to a therapy for aggressive NHL is assessed according to thecriteria described in Cheson et al. (2014) “Recommendations for InitialEvaluation, Staging and Response Assessment of Hodgkin and Non-HodgkinLymphoma: The Lugano Classification.” J. Clin Oncol. 32: 3059-3067.

In some embodiments, the individual was refractory to or had relapsedfollowing treatment with at least one prior therapy (e.g., at least oneprior standard approved therapy, at least two prior standard approvedtherapies, at least three standard approved therapies etc.) foraggressive NHL. Standard therapies for DLBCL (e.g., de novo ortransformed DLBCL) and MCL include, but are not limited to, e.g.,rituximab, RCHP (i.e., rituximab, cyclophosphamide, doxorubicin, andprednisone); R-CHOP (i.e., rituximab, cyclophosphamide, doxorubicin,vincristine, and prednisone); R-CHOEP (i.e., rituximab,cyclophosphamide, doxorubicin, vincristine, etoposide, and prednisonethat is typically administered in 21-day cycles for 6 cycles); EPOCH-R(i.e., rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide,and prednisone that is typically administered as a continuous infusionover 4 days); R-GCVP (i.e., rituximab, gemciabine, cyclopiosphamide,vMcristine, and prednisolone); R-CEPP (i.e., rituximab,cyclphospharnide, etoposide, procarbazine, and prednisone-RCEOP (i.e.,rituximab, cyclophosphamide, epirubicin, vincristine, and prednisone):R-CVP (i.e., rjiximab, cyclophosphammid, vincristine, and prednisone);rituximab and bendanustine; rituximab and lenalidomide; DIHAP i.e.,dexamethasone, high-dose cytarabine and cisplatin); RDHAP (i.e., DHAP incombination with rituximab); ICE (i.e., ifosfamide, carboplatin, andetoposide); RICE (i.e., ICE in combination with rituximab); DICE (i.e.,ICE in combination with dexamethasone); DICE and mesna; BEAM (i.e.,carmustine, etoposide, cytarabine, and melphalan); R-BEAM (i.e., BEAM incombination with rituximab); ESHAP (i.e., etoposide, solumedrol,high-dose cytarabine, and cisplatin); R-ESHAP (i.e., ESHAP incombination with rituximab); MIME (i.e.,methyl-glyoxal-bis(guanylhydrazone), ifosfamide, methotrexate, andetoposide); parsaclisib (also known as INCB050465); MATRIX (i.e.,methotrexate, cytarabine, thiotepa, and rituximab); hyper-CVAD or HCVAD(i.e., hyper-fractionated cyclophosphamide, vincristine, doxorubicin,and dexamethasone) RHCVAD (i.e., HCVAD in combination with rituximab);RHCVAD/MA (i.e., RHCVAD alternating with methotrexate and cytarabine);DHAP (i.e., dexamethasone, cisplatin, and cytarabine); R-DHAP (i.e.,DHAP in combination with rituximab); ibrutinib; rituximab, obinituzumab,CVAD (i.e., cyclophosphamide, doxorubicin, vincristine, andprednisolone); RCVAD (i.e., CVAD in combination with rituximab); GemOx(i.e., gemcitabine and oxaliplatin); R-GemOx (i.e., GemOx in combinationwith rituximab); DHAX (i.e., dexamethasone, cytarabine, andoxaliplatin); R-DHAX (i.e., DHAX in combination with rituximab); GIFOX(i.e., gemcitabine, ifosfamide, and oxaliplatin); RGIFOX (i.e., GIFOX incombination with rituximab); bortezomib and GIFOX; ASCT (i.e.,autologous stem cell transplantation) HD-ASCT (i.e., ASCT in combinationwith high dose therapy, e.g., high-dose chemotherapy); CAR T-celltherapy (e.g., tisagenlecleucel or axicabtagene); brentuximab vedotin,and lenalidomide. In some embodiments, the prior therapy for aggressiveNHL comprised any two or more of the preceding treatments (giventogether in a single treatment regimen, or given in separate treatmentregimens). In some embodiments, there are no available treatment options(e.g., curative treatment options) for the individual with aggressiveNHL (e.g., relapsed/refractory aggressive NHL). In some embodiments, theindividual has DLBCL (e.g., de novo DLBCL or transformed DLBCL) or MCLfor which no curative therapy is available. In some embodiments, theindividual has DLBCL (e.g., de novo DLBCL or transformed DLBCL) or MCLthat has relapsed following or has been refractory to standard approvedtherapies (e.g., curative therapies).

(ii) Indolent Lymphoma

In some embodiments, provided is a method of treating indolent lymphomain an individual (e.g., a human individual) that comprises administeringto the individual an effective amount of (a) a polypeptide (e.g., fusionpolypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1domain variant described herein) and an Fc domain variant (e.g., an Fcdomain variant described herein) and (b) a therapeutic anti-CD20antibody, wherein the indolent lymphoma is relapsed and/or refractoryindolent lymphoma (e.g., wherein the individual has relapsed during orfollowing at least one prior treatment, e.g., a standard approvedtherapy, for indolent lymphoma and/or or has been refractory to at leastone prior treatment, e.g., a standard approved therapy, for indolentlymphoma. In some embodiments, the individual has relapsed during orafter more than one standard approved therapy (e.g., 2, 3, or morestandard therapies) for indolent lymphoma and/or is refractory to morethan one standard therapy (e.g., 2, 3, or more standard therapies, e.g.,curative therapies) for indolent lymphoma In some embodiments, thepolypeptide (e.g., fusion polypeptide) comprises a SIRPα D1 domainvariant that comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85. In some embodiments, the Fc domain variant is (i) a humanIgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations,wherein numbering is according to the EU index of Kabat; (ii) a humanIgG2 Fc region comprising A330S, P331S, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (iii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations,wherein numbering is according to the EU index of Kabat; or (iv) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, deG236, and N297Amutations, wherein numbering is according to the EU index of Kabat. Insome embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises a SIRPα D1 domain variant thatcomprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. Insome embodiments the polypeptide (e.g., fusion polypeptide) administeredto the individual comprises an Fc domain variant that is a human IgG1 Fcregion comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat. In some embodiments,the Fe domain variant comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the polypeptide (e.g., fusion polypeptide)administered to the individual comprises the amino acid sequence of SEQID NO: 136 or SEQ ID NO: 135. In some embodiments the polypeptide (e.g.,fusion polypeptide) forms a homodimer. In some embodiments, thetherapeutic anti-CD20 antibody is rituximab.

In some embodiments, the rituximab is administered subcutaneously. Insome embodiments, the rituximab is administered via intravenousinfusion. In some embodiments, the rituximab is administered accordingto its label instructions. In some embodiments, the rituximab isadministered to the individual (e.g., via intravenous infusion) at adose of about 375 mg/m². In some embodiments, the first four doses (i.e.doses 1-4) of rituximab are administered to the individual (e.g., at adose of about 375 mg/m²) once a week (e.g., once every 7 days or “qw”)for the first four weeks (e.g., 28 days) of treatment, and the nexteight doses (i.e., doses 5-12) are administered to the individual onceevery four weeks (e.g., every 28 days or “q4w”). In some embodiments,the first four doses (i.e. doses 1-4) of rituximab are administered tothe individual (e.g., at a dose of about 375 mg/m²) once a week (e.g.,once every 7 days or “qw”) for the first four weeks (e.g., 28 days) oftreatment, and the next four doses (i.e., doses 5-8) are administered tothe individual once every four weeks (e.g., every 28 days or “q4w”). Insome embodiments, dose modifications of rituximab are made according tothe local package insert. Complete information about rituximabpreparation, dispensing, dosage, and administration schedule can befound in the local package insert (for the United States, see, e.g.,www(dot)accessdata(dot)fda(dot)gov/drugsatfda_docs/label/2012/103705s5367s53881bl(dot)pdf,for Europe, see, e.g.,www(dot)ema(dot)europa(dot)eu/en/documents/product-information/mabthera-epar-product-information_en(dot)pdf).In some embodiments, the polypeptide (e.g., fusion polypeptide) isadministered (e.g., via intravenous infusion) to the individual weekly(i.e., once every 7 days or “qw”), e.g., at a dose of 10.0 mg/kg or 15.0mg/kg.

In some embodiments, the indolent lymphoma is an indolent non-Hodgkinlymphoma (NHL). In some embodiments, the indolent NHL is marginal zonelymphoma (MZL). In some embodiments, the indolent NHL is follicularlymphoma (FL). Details regarding the diagnosis and classification ofmarginal zone lymphoma and follicular lymphoma (as well as DLBCL, andmantle cell lymphoma) are provided in, e.g., Ayyappan et al. (2018) CurrOncol Rep. 20(4): 33; Dreyling et al. (2013) ESMO Consensus Guidelines:Marginal Cell Lymphoma, Mantle Cell Lymphoma, Peripheral T-cellLymphoma.” Ann Oncol. 24(4): 857-877; Vose, J M (2017) “Mantle celllymphoma: 2017 update on diagnosis, risk-stratification, and clinicalmanagement.” Am J Hematol. 92(8): 806-813; Ciobanu et al. (2013)“Indolent Lymphoma: Diagnosis and Prognosis in Medical Practice.”Maedica (Buchar) 8(4) 338-342, and others.

In some embodiments, the individual is considered to have relapsedfollowing a prior therapy for indolent lymphoma (e.g., indolent NHL) ifthe individual achieved a therapeutic response of least stable disease(SD) to the prior therapy, but stopped responding (e.g., demonstrateddisease progression) within about any one of, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 months following the cessation of the prior therapy.In some embodiments, the individual is considered to be refractory to aprior therapy for indolent lymphoma (e.g., indolent NHL) if theindividual was unresponsive to the prior therapy (e.g., failed toachieve a therapeutic response of at least stable disease (SD) during orfollowing the prior therapy). In some embodiments, the therapeuticresponse to therapy for indolent lymphoma (e.g., indolent NHL) isassessed according to the criteria described in Cheson et al. (2014)“Recommendations for Initial Evaluation, Staging and Response Assessmentof Hodgkin and Non-Hodgkin Lymphoma: The Lugano Classification.” J. ClinOncol. 32: 3059-3067.

In some embodiments, the individual was refractory to or had relapsedfollowing treatment with at least one prior therapy (e.g., at least oneprior standard approved therapy, at least two prior standard approvedtherapies, at least three standard approved therapies etc.) for indolentlymphoma (e.g., indolent NHL). Standard therapies for indolent lymphoma(e.g., indolent NHL, such as marginal zone lymphoma or follicularlymphoma) include, but are not limited to, e.g., the standard therapiesfor aggressive NHL (e.g., DLBCL or mantle cell lymphoma), which aredescribed in detail elsewhere herein. Other standard therapies forindolent lymphoma (e.g., indolent NHL) include, but are not limited to,e.g., fludarabine, FR (i.e., fludarabine and rituximab); FCR (i.e., FRin combination with cyclophosphamide); FCM (i.e., fludarabine,cyclophosphamide, mitoxantrone); FCMR (i.e., FCM in combination withrituximab); ibritumomab tiuxetan; tositumomab; vorinostat; everolimus;bortezomib; navitoclax (also known as ABT-263); high dose therapy (HDT);autologous stem cell transplantation; and allogenic stem celltransplantation. In some embodiments, the prior therapy for indolentlymphoma (e.g., indolent NHL) comprised any two or more of the precedingstandard therapies (including therapies for aggressive NHL, which aredescribed elsewhere herein). In some embodiments, the two or morestandard therapies for indolent lymphoma (e.g., indolent NHL) (includingstandard treatments for aggressive NHL) were given together in a singletreatment regimen. In some embodiments, the two or more standardtherapies for indolent NHL (including standard treatment for aggressiveNHL) were given in separate treatment regimens. In some embodiments,there are no available treatment options (e.g., curative treatmentoptions) for the individual with indolent lymphoma (e.g., indolent NHL)(e.g., relapsed/refractory indolent NHL).

In some embodiments of any of the methods of treatment provided herein,the fusion polypeptide is supplied for use (e.g., intravenousadministration) in a 100 mg/5 ml Type I clear glass vial sealed with a20 mm Teflon coated rubber septum stopper and aluminum seal. In someembodiments, the fusion polypeptide is supplied for use (e.g.,intravenous administration) in a 400 mg/20 ml Type I clear glass vialsealed with a 20 mm Teflon coated rubber septum stopper and aluminumseal. In some embodiments, the fusion polypeptide is stored in itsoriginal container at 2-8° C. (36-46° F.) until use (e.g., intravenousadministration).

In some embodiments of any of the methods of treatment described herein,the polypeptide (e.g., fusion polypeptide comprising a SIRPα D1 domainvariant and an Fc domain variant) is administered subcutaneously. Insome embodiments, the polypeptide (e.g., fusion polypeptide) isadministered via intravenous infusion. In some embodiments, thepolypeptide (e.g., fusion polypeptide) is administered (e.g., viaintravenous infusion) to the individual (e.g., human individual) at adose of 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10.0 mg/kg, 15.0 mg/kg, or 30.0mg/kg, including any range in between these values. In some embodiments,the polypeptide (e.g., fusion polypeptide) is administered (e.g., viaintravenous infusion) to the individual weekly (i.e., once every 7 daysor “qw”), e.g., at a dose of 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10.0mg/kg, 15.0 mg/kg, or 30.0 mg/kg, including any range in between thesevalues. In some embodiments, the polypeptide (e.g., fusion polypeptide)is administered to the individual (e.g., via intravenous infusion) everyother week (i.e., once every 14 days or “q2w” or “QoW”), e.g., at a doseof 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10.0 mg/kg, 15.0 mg/kg, or 30.0mg/kg, including any range in between these values. In some embodiments,on the days when the dosing schedules of the polypeptide (e.g., fusionpolypeptide) and the therapeutic antibody (e.g., the anti-PD1 antibody(pembrolizumab), the anti-HER2 antibody (trastuzumab), or the anti-CD20antibody (rituximab)) coincide, the polypeptide and the therapeuticantibody are administered sequentially. In some embodiments, thepolypeptide (e.g., fusion polypeptide) is administered prior (e.g.,about 30 minutes prior) to the therapeutic antibody. In someembodiments, in the event of a missed dose of the polypeptide, thetherapeutic antibody is administered about 24 hours after the misseddose.

In some embodiments, the therapeutic response of an individual havingNSCLC, HNSCC, gastric cancer or GEJ cancer to a method of treatmentprovided herein is assessed according to the RECIST version 1.1criteria, e.g., as described in Therasse et al. (2000) J Natl CancerInst. 92: 205-216; Eisenhauer et al. (2009) Eur J. Cancer. 45: 229-247,the immune-related response criteria derived from RECIST 1.1 (irRECIST),e.g., as adapted from Nishino, et al. (2013) “Developing a CommonLanguage for Tumor Response to Immunotherapy: Immune-Related ResponseCriteria Using Unidimensional Measurements.” Clinical Cancer Research19(14):3936-43. In some embodiments, the therapeutic response of anindividual having aggressive lymphoma (e.g., aggressive NHL such asDLBCL or MCL) to a method of treatment provided herein is assessedaccording to the Lugano criteria, e.g., as described in Cheson et al.(2014) “Recommendations for Initial Evaluation, Staging and ResponseAssessment of Hodgkin and Non-Hodgkin Lymphoma: The LuganoClassification.” J. Clin Oncol. 32: 3059-3067. In some embodiments, thetherapeutic response of an individual having indolent lymphoma (e.g.,indolent NHL such as FL or MZL) to a method of treatment provided hereinis assessed according to the Lugano criteria (see Cheson et al 2014).

In some embodiments, the individual receiving treatment for HNSCC,NSCLC, gastric cancer, or GEJ cancer has at least one measurable lesionas defined by RECIST version 1.1 criteria, e.g., as described inTherasse et al. (2000) J Natl Cancer Inst. 92: 205-216; Eisenhauer etal. (2009) Eur J Cancer. 45: 229-247. In some embodiments, theindividual receiving treatment for lymphoma (e.g., aggressive lymphoma(such as DLBCL or MCL) or indolent lymphoma (such as FL or MZL) has atleast one measurable lesion as defined by Lugano criteria e.g., asdescribed in Cheson et al. (2014) “Recommendations for InitialEvaluation, Staging and Response Assessment of Hodgkin and Non-HodgkinLymphoma: The Lugano Classification.” J. Clin Oncol. 32: 3059-3067.

In some embodiments, the individual receiving treatment according to amethod herein has adequate bone marrow function, renal function, liverfunction, and cardiac function. In some embodiments, the individual hasan Eastern Cooperative Oncology Group (ECOG) Performance Status (PS)score of 0 or 1 (see, e.g.,www(dot)npcrc(dot)org/files/news/ECOG)_performance_status(dot)pdf). Insome embodiments, the individual does not have symptomatic centralnervous system (CNS) metastases or leptomeningeal disease requiringsteroids. In some embodiments, the individual receiving treatment forlung cancer according to a method herein (e.g., NSCLC) does not have ALKor EGFR genomic tumor aberrations. In some embodiments, an individualreceiving treatment according to a method herein does not have a historyof (non-infectious) pneumonitis that required steroids or has currentpneumonitis. In some embodiments, the individual receiving treatmentaccording to a method herein does not have high grade lymphoma (e.g.,Burkitts lymphoma, lymphoblastic lymphoma, or Richter's transformation),chronic lymphocytic leukemia, or plasma cell leukemia. In someembodiments, the individual has not undergone high-dose chemotherapyrequiring allogeneic stem cell rescue. In some embodiments, theindividual receiving treatment for lung cancer (e.g., NSCLC), head andneck cancer (e.g., HNSCC), or gastric/GEJ cancer (e.g., HER2-positivegastric/GEJ adenocarcinoma) according to a method herein has notundergone prior irradiation to >25% of the bone marrow. In someembodiments, the individual has not received radiotherapy within 2 weeksof start of treatment. In some embodiments, the individual has notreceived prior treatment with any anti-CD47 or anti-SIRPα agent. In someembodiments, the individual has not received systemic anti-cancertherapy within 4 weeks of starting treatment (6 weeks for mitomycin C ornitrosoureas). In some embodiments, the individual does not have anintolerance to or has not had a severe allergic or anaphylactic reactionto antibodies or infused therapeutic protein(s) or any excipients in theformulation(s) comprising the therapeutic protein. In some embodiments,the individual has not discontinued treatment due to a Grade 3 or higherimmune-related adverse event (AE) from prior therapy with an anti-PD-1,anti-PD-L1, or anti PD-L2 agent or with an agent aiming to modulateanother immune cell target (e.g. CTLA-1, OX40, 41BB, etc.). In someembodiments, the individual has not received experimental antibodies orlive vaccines (e.g., including, but not limited to vaccines for measles,mumps, rubella, varicella/zoster, yellow fever, rabies, BacillusCalmette-Guérin (BCG), typhoid, and intranasal influenza vaccines). Insome embodiments, the individual is not undergoing current activetherapy for the primary diagnosis (e.g., lung cancer (NSCLC), head andneck cancer (HNSCC), gastric/GEJ cancer (HER2-positive gastric/GEJadenocarcinoma), aggressive lymphoma (de novo DLBCL or transformed DLBCLor mantle cell lymphoma), or indolent lymphoma (e.g., indolent NHL, suchas marginal zone lymphoma or follicular lymphoma). In some embodiments,the individual has not received a blood product transfusion within 14days of the start of treatment. In some embodiments, the individual doesnot have a history of active autoimmune disorders (including but notlimited to, e.g., Crohn's Disease, rheumatoid arthritis, scleroderma,systemic lupus erythematosus, Grave's disease, autoimmune hemolyticanemia, autoimmune thrombocytopenia) and other conditions thatcompromise or impair the immune system (other thanhypogammaglobulinemia). In some embodiments, the individual does nothave an active, uncontrolled, clinically significant bacterial, fungal,or viral infection, including hepatitis B (HBV), hepatitis C (HCV),known human immunodeficiency virus (HIV) or acquired immunodeficiencysyndrome (AIDS)-related illness. In some embodiments, the individualdoes not have active graft versus host disease (GVHD) or is notundergoing immunosuppression therapy for GVHD. In some embodiments, theindividual has not had any of the following in the previous 12 months:myocardial infarction, severe/unstable angina, coronary/peripheralartery bypass graft, symptomatic congestive heart failure,cerebrovascular accident, transient ischemic attack, deep venousthrombosis, or symptomatic pulmonary embolism. In some embodiments, theindividual has not been diagnosed with any other malignancy within thelast 3 years prior to the start of treatment, except for, e.g.,adequately treated non-melanomatous skin cancer, or carcinoma in situ(e.g., breast carcinoma, cervical cancer in situ) that have undergonepotentially curative therapy.

Kits and Articles of Manufacture

In another embodiment of the invention, an article of manufacture or akit is provided comprising a polypeptide (e.g., a fusion polypeptidedescribed herein) comprising a SIRPα D1 domain variant and an Fc domainvariant. In some embodiments, the SIRPα D1 domain variant comprises theamino acid sequence selected from the group consisting of: SEQ ID NO:81, SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) ahuman IgG1 Fc region comprising L234A, L235A, G237A, and N297Amutations, wherein numbering is according to the EU index of Kabat; (ii)a human IgG2 Fc region comprising A330S, P331S, and N297A mutations,wherein numbering is according to the EU index of Kabat; (iii) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236mutations, wherein numbering is according to the EU index of Kabat; or(iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,deG236, and N297A mutations, wherein numbering is according to the EUindex of Kabat. In some embodiments, the Fc domain variant comprises theamino acid sequence of SEQ ID NO: 91. In some embodiments thepolypeptide comprises the amino acid sequence of SEQ ID NO: 135 or SEQID NO: 136. In some embodiments, the polypeptide forms a homodimer. Insome embodiments, the kit or article of manufacture is for use accordingto a method of treatment provided herein.

In some embodiments, the kit or article of manufacture further comprisesan anti-PD1 antibody. In some embodiments, the anti-PD1 antibody ispembrolizumab. In some embodiments, the kit comprises a package insertor label with instructions for using the polypeptide (e.g., fusionpolypeptide) in conjunction with the anti-PD1 antibody (e.g.,pembrolizumab) to treat or delay progression of lung cancer (e.g.,NSCLC, including metastatic NSCLC) in an individual according to amethod herein. In some embodiments, the kit comprises a package insertor label with instructions for using the polypeptide (e.g., fusionpolypeptide) in conjunction with the anti-PD1 antibody (e.g.,pembrolizumab) to treat or delay progression of lung cancer (e.g.,NSCLC, including metastatic NSCLC) in an individual has received priortherapy for NSCLC. In some embodiments, the individual has progressed(e.g., demonstrated disease progression) during (or following) a priortherapy (e.g., prior immune checkpoint inhibitor therapy) for lungcancer. In some embodiments, the individual has a PD-L1 tumor proportionscore (TPS) of less than 50%. In some embodiments, the kit comprises apackage insert or label with instructions for using the polypeptide(e.g., fusion polypeptide) in conjunction with the anti-PD1 antibody(e.g., pembrolizumab) to treat or delay progression of HNSCC in anindividual according to a method herein. In some embodiments, the kitcomprises a package insert or label with instructions for using thepolypeptide (e.g., fusion polypeptide) in conjunction with the anti-PD1antibody (e.g., pembrolizumab) to treat or delay progression of HNSCC inan individual who has received prior immune checkpoint inhibitor therapy(e.g., for HNSCC). In some embodiments, the kit comprises a packageinsert or label with instructions for using the polypeptide (e.g.,fusion polypeptide) in conjunction with the anti-PD1 antibody (e.g.,pembrolizumab) to treat or delay progression of HNSCC in an individualwho has not received prior immune checkpoint inhibitor therapy (e.g.,for HNSCC). In some embodiments, the kit comprises a package insert orlabel with instructions for using the polypeptide (e.g., fusionpolypeptide) in conjunction with the anti-PD1 antibody (e.g.,pembrolizumab) to treat or delay progression of head and neck cancer(e.g., HNSCC, including metastatic HNSCC) in an individual who hasprogressed (e.g., demonstrated disease progression) on (or following) aprior platinum therapy (e.g., a platinum-containing therapy). In someembodiments, the kit or article of manufacture further comprisesinstructions for administering the pembrolizumab at a dose of 200 mgevery 3 weeks (Q3W) by IV infusion. In some embodiments, the kit orarticle of manufacture further comprises instructions for administeringthe polypeptide (e.g., fusion polypeptide) at a dose of 10 mg/kg everyweek (QW) by IV infusion.

In some embodiments, the kit or article of manufacture further comprisesan anti-HER2 antibody. In some embodiments, the anti-HER2 antibody istrastuzumab. In some embodiments, the kit comprises a package insert orlabel with instructions for using the polypeptide (e.g., fusionpolypeptide) in conjunction with the anti-HER2 antibody (e.g.,trastuzumab) to treat or delay progression of HER2-positive gastric orHER2-positive GEJ cancer (e.g., HER2-positive gastric adenocarcinoma orHER2-positive GEJ adenocarcinoma) in an individual according to a methodprovided herein. In some embodiments, the kit comprises a package insertor label with instructions for using the polypeptide (e.g., fusionpolypeptide) in conjunction with the anti-HER2 antibody (e.g.,trastuzumab) to treat or delay progression of HER2-positive gastric orHER2-positive GEJ cancer (e.g., HER2-positive gastric adenocarcinoma orHER2-positive GEJ adenocarcinoma) in an individual who has progressed(e.g., demonstrated disease progression) while on (or following) a priortherapy for gastric or GEJ cancer. In some embodiments the prior therapycomprised anti-HER2 antibody and/or a prior fluoropyrimidine-basedtherapy. In some embodiments, the kit or article of manufacture furthercomprises instructions for administering the trastuzumab via intravenousinfusion once every three weeks (q3W), wherein the initial dose oftrastuzumab is 8 mg/kg and each subsequent dose of trastuzumab (i.e.,following the initial dose) is 6 mg/kg. In some embodiments, the kit orarticle of manufacture further comprises instructions for administeringthe polypeptide (e.g., fusion polypeptide) at a dose of 10 mg/kg everyweek (QW) by IV infusion.

In some embodiments, the kit or article of manufacture further comprisesan anti-CD20 antibody. In some embodiments, the anti-CD20 antibody isrituximab. In some embodiments, the kit comprises a package insert orlabel with instructions for using the polypeptide (e.g., fusionpolypeptide) in conjunction with the anti-CD20 antibody (e.g.,rituximab) to treat or delay progression of aggressive non-Hodgkinlymphoma or “NHL” (e.g., de novo DLBCL or transformed DLBCL, or mantlecell lymphoma) in an individual according to a method herein. In someembodiments, the kit comprises a package insert or label withinstructions for using the polypeptide (e.g., fusion polypeptide) inconjunction with the anti-CD20 antibody (e.g., rituximab) to treat ordelay progression of aggressive non-Hodgkin lymphoma or “NHL” (e.g., denovo DLBCL or transformed DLBCL, or mantle cell lymphoma) in anindividual who has relapsed or was refractory to prior therapy (e.g.,prior standard therapy/curative therapy) for aggressive NHL, or in anindividual for whom there is no available therapy (e.g., curativetherapy) for aggressive NHL. In some embodiments, the kit comprises apackage insert or label with instructions for using the polypeptide(e.g., fusion polypeptide) in conjunction with the anti-CD20 antibody(e.g., rituximab) to treat or delay progression of indolent lymphoma(e.g., indolent NHL, such as marginal zone lymphoma or follicularlymphoma) in an individual according to a method herein. In someembodiments, the kit comprises a package insert or label withinstructions for using the polypeptide (e.g., fusion polypeptide) inconjunction with the anti-CD20 antibody (e.g., rituximab) to treat ordelay progression of indolent lymphoma (e.g., indolent NHL, such asmarginal zone lymphoma or follicular lymphoma) in an individual who hasrelapsed or was refractory to prior therapy (e.g., prior standardtherapy, e.g., curative therapy) for indolent lymphoma (e.g., indolentNHL), or in an individual for whom there is no available therapy (e.g.,curative therapy) for indolent lymphoma (e.g., indolent NHL). In someembodiments, the kit or article of manufacture further comprisesinstructions for administering the rituximab to the individual at a doseof 375 mg/m² once a week (e.g., once every 7 days or “qw”) for the firstfour weeks (e.g., 28 days) of treatment, and then administering therituximab to the individual at a dose of 375 mg/m² once every four weeks(e.g., every 28 days or “q4w”) for up to four additional doses followingthe first four doses, or for up to 8 additional doses following thefirst four doses. In some embodiments, the kit or article of manufacturefurther comprises instructions for administering the polypeptide (e.g.,fusion polypeptide) at a dose of 10 mg/kg or 15 mg/kg every week (QW) byIV infusion.

In some embodiments of any of the kits or articles of manufactureprovided herein, the polypeptide (e.g., fusion polypeptide) and thetherapeutic antibody (e.g., pembrolizumab, trastuzumab, or rituximab)are in the same container or separate containers. Suitable containersinclude, for example, bottles, vials, bags and syringes. The containermay be formed from a variety of materials such as glass, plastic (suchas polyvinyl chloride or polyolefin), or metal alloy (such as stainlesssteel or hastelloy). In some embodiments, the container holds theformulation and the label on, or associated with, the container mayindicate directions for use. The article of manufacture or kit mayfurther include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use. In someembodiments, the article of manufacture further includes one or more ofanother agent (e.g., a chemotherapeutic agent, and anti-neoplasticagent). Suitable containers for the one or more agents include, forexample, bottles, vials, bags and syringes.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

EXAMPLES Example 1: A Phase 1 Study of Drug A in Combination withEstablished Anticancer Antibodies in Patients with Advanced Malignancies

This Example describes a Phase 1 clinical study that evaluated thesafety, efficacy, pharmacodynamics (PD) and pharmacokinetics (PK) ofDrug A in combination with pembrolizumab, trastuzumab, or rituximab forpatients with advanced malignancies. Drug A is a fusion proteinconsisting of a high affinity CD47-binding SIRPα D1 domain variant fusedto a human immunoglobulin Fc domain variant that is modified toeliminate binding to Fc gamma receptors (FIG. 1).

Study Objectives

Primary Objective

The primary objective of this study was to evaluate the safety andtolerability of Drug A administered once every week and/or every 2 weeksin combination with pembrolizumab, trastuzumab, or rituximab in patientswith advanced malignancies including non-small cell lung cancer (NSCLC),head and neck squamous cell carcinoma (HNSCC), HER2-overexpressinggastric cancer, and non-Hodgkin lymphoma (NHL).

Secondary Objectives

The secondary objectives of this study were:

-   -   To evaluate the anti-tumor effect of Drug A in combination with        anti-cancer therapeutics in patients with advanced malignancies.    -   To evaluate the overall safety profile of Drug A in combination        with other anti-cancer therapeutics.    -   To characterize the maximum tolerated dose (MTD)/optimal        biological dose (OBD) of Drug A in combination with anti-cancer        therapeutic agents.    -   To characterize the single and multiple dose pharmacokinetics of        Drug A in combination with the anti-cancer therapeutics        pembrolizumab, trastuzumab or rituximab.    -   To evaluate the immunogenicity of Drug A.

Exploratory Objectives

The exploratory objective of this study was to explore thepharmacodynamic effect of Drug A in combination with anti-cancertherapeutics in patients with advanced malignancies.

Patients

Inclusion Criteria

All patients met the following criteria for the dose escalation phase ofthe study:

-   -   Histological or cytological diagnosis of advanced/metastatic        solid tumor malignancy or relapsed/refractory CD20⁺ B Cell non        Hodgkin lymphoma that:        -   Was resistant to standard therapy or for which no curative            therapy was available; or        -   Met any criteria (a-d) for dose expansion described below    -   Lesions could be measurable or non-measurable.

All patients met the following criteria for the dose expansion phase ofthe study:

-   -   a) Locally advanced or metastatic NSCLC (TPS ≥1%) which had        progressed on prior checkpoint therapy, OR Locally advanced or        metastatic NSCLC (TPS <50%) that had progressed following        systemic therapy for their metastatic disease; patients were        suitable for treatment with pembrolizumab; Or    -   b) Recurrent or metastatic HNSCC with disease progression on or        after platinum-containing chemotherapy and were suitable for        treatment with pembrolizumab; Or    -   c) HER2 overexpressing metastatic gastric or gastroesophageal        junction (GEJ) adenocarcinoma that had progressed following        systemic therapy with a fluoropyrimidine-containing regimen        and/or therapy with an anti-HER2 antibody for their metastatic        disease and were suitable for treatment with trastuzumab;    -   d) Relapsed or refractory, de novo or transformed diffuse large        B-cell lymphoma (DLBCL), or mantle cell lymphoma for which no        curative therapy was available; OR indolent lymphoma (Marginal        zone, follicular,) that was relapsed or refractory to standard        approved therapies.    -   Patients had at least one measurable lesion as defined by RECIST        version 1.1 or Lugano criteria (2014).

In addition, all patients met the following criteria:

-   -   Adequate Bone Marrow Function, including:        -   Absolute Neutrophil Count (ANC) ≥1,500/mm³ (≥1.5×10⁹/L); Non            Hodgkin lymphoma, only, ANC ≥1,000/mm³ (≥1.0×10⁹L);        -   Platelets ≥75,000/mm³ (≥75×10⁹/L); Non Hodgkin lymphoma            only: Platelets ≥50,000/mm³ (≥50×10⁹);        -   Hemoglobin ≥9 g/dL (≥90 g/L); Non Hodgkin lymphoma only:            Hemoglobin ≥8 g/dL (≥80 g/L).    -   Adequate Renal Function, including:        -   Serum creatinine ≤1.5× upper limit of normal (ULN) or            estimated creatinine clearance ≥60 mL/min as calculated            using the method standard for the institution.    -   Adequate Liver Function, including:        -   Total serum bilirubin ≤1.5×ULN (≤3.0×ULN if the patient had            documented Gilbert syndrome);        -   Aspartate and Alanine transaminase (AST and ALT)≤3.0×ULN;            ≤5.0×ULN if there was liver involvement secondary to tumor;        -   Alkaline phosphatase ≤2.5×ULN (≤5.0×ULN if bone or liver            metastasis).    -   QT interval corrected for heart rate Fridericia's (QTcF)        interval of ≤480 msec (based upon mean value from triplicate        ECGs).    -   Age ≥18 years.    -   Eastern Cooperative Oncology Group (ECOG) Performance Status        (PS) was 0 or 1.    -   Resolved acute effects of any prior therapy to baseline severity        or Grade ≤1 (NCI CTCAE v.4.03) except for AEs not constituting a        safety risk by Investigator judgment.    -   Available archival (or fresh) metastatic biopsy sample prior to        study entry (expansion phase only).    -   Serum pregnancy test (for females of childbearing potential)        negative at screening.

Exclusion Criteria

Patients with any of the following characteristics were not included inthis study:

-   -   Patients with known symptomatic CNS metastases or leptomeningeal        disease requiring steroids. Patients with previously diagnosed        brain metastases were eligible if they completed their treatment        and recovered from the acute effects of radiation therapy or        surgery prior to study entry, discontinued corticosteroid        treatment for these metastases and were clinically stable off        anticonvulsants for at least 4 weeks and were neurologically        stable before enrollment.    -   Patients with ALK or EGFR genomic tumor aberrations (NSCLC, Part        2 expansion only), or any patient with a history of        (non-infectious) pneumonitis that required steroids or had        current pneumonitis.    -   Patients with high grade lymphoma (including Burkitts lymphoma,        lymphoblastic lymphoma, Richter's transformation), CLL or plasma        cell leukemia    -   Previous high-dose chemotherapy requiring allogeneic stem cell        rescue.    -   Prior irradiation to >25% of the bone marrow (non-lymphoma        patients only), or any patient who received prior radiotherapy        within 2 weeks of start of study treatment. Note: Participants        must have recovered from all radiation-related toxicities, did        not require corticosteroids, and not have had radiation        pneumonitis. A 1-week washout was permitted for palliative        radiation (≤2 weeks of radiotherapy) to non-CNS disease.    -   Prior treatment with any anti-CD47 or anti-SIRPα agent.    -   Systemic anti-cancer therapy within 4 weeks of starting study        treatment (6 weeks for mitomycin C or nitrosoureas). If systemic        anti-cancer therapy was given within 4 weeks, patient was        included if 4-5 times elimination half-life of the drug had        passed.    -   Prior treatment with (Expansion phase only):        -   A PD-1 or PD-L1 inhibitor (NSCLC; HNSCC) for metastatic            disease with disease progression within 8 weeks of            initiation.        -   Trastuzumab (Gastric/GEJ Cancer) for metastatic disease with            disease progression as the best response.    -   Patients with intolerance to or who had a severe allergic or        anaphylactic reaction to antibodies or infused therapeutic        proteins, or patients who had a severe allergic or anaphylactic        reaction to any of the substances included in the study drug        (including excipients); or who discontinued treatment due to a        Grade 3 or higher immune related AE from prior therapy with an        anti-PD-1, anti-PD-L1, or anti PD-L2 agent or with an agent        aiming to modulate another immune cell target (e.g., CTLA-1,        OX40, 41BB, etc.).    -   Any experimental antibodies or live vaccines in the last 28 days        prior to the first dose of study drug. Live attenuated vaccines        were not allowed.    -   Current active therapy for the primary diagnosis.    -   Blood product transfusions within 14 days of Cycle 1 Day 1.    -   History of (or active) autoimmune disorders (including but not        limited to: Crohn's Disease, rheumatoid arthritis, scleroderma,        systemic lupus erythematosus, Grave's disease) and other        conditions that compromise or impair the immune system (with the        exception of hypogammaglobulinemia).    -   History of autoimmune hemolytic anemia or autoimmune        thrombocytopenia.    -   Patients with active, uncontrolled, clinically significant        bacterial, fungal, or viral infection, including hepatitis B        (HBV), hepatitis C (HCV), known human immunodeficiency virus        (HV) or acquired immunodeficiency syndrome (AIDS)-related        illness.    -   Patients with active graft versus host disease (GVHD) or ongoing        immunosuppression for GVHD.    -   Any of the following in the previous 12 months: myocardial        infarction, severe/unstable angina, coronary/peripheral artery        bypass graft, symptomatic congestive heart failure,        cerebrovascular accident, transient ischemic attack, deep venous        thrombosis, or symptomatic pulmonary embolism.    -   Current active treatment in another interventional therapeutic        clinical study.    -   Diagnosis of any other malignancy within the last 3 years prior        to enrollment except for adequately treated non-melanomatous        skin cancer, or carcinoma in situ (e.g., breast carcinoma,        cervical cancer in situ) that underwent potentially curative        therapy.    -   Other severe acute or chronic medical or psychiatric condition,        including recent (within the past year) or active suicidal        ideation or behavior, or laboratory abnormality that increased        the risk associated with study participation or investigational        product administration or interfered with the interpretation of        study results and, in the judgment of the Investigator, made the        patient inappropriate for entry into this study.    -   Males and females of childbearing potential not using highly        effective contraception or not having agreed to continue highly        effective contraception for at least 90 days after last dose of        investigational product.    -   Patients who were pregnant or breastfeeding.

Study Treatment

As shown in FIG. 2, this study had three treatment arms(DrugA+pembrolizumab; Drug A+trastuzumab; DrugA+rituximab). The studyincluded an initial dose escalation portion followed by a dose expansionportion. Each dose escalation group had approximately 12 advancedmalignancy patients. Each expansion group had approximately 20-40patients at multiple sites.

Drug A was administered weekly (or every 2 weeks) as an IV infusion overapproximately 60 minutes on an outpatient basis. The use of an infusionpump was the preferred method of administration to ensure accuratedelivery of the investigational product, but gravity drips were allowed.

Drug A was supplied in either a 100 mg/5 mL or 400 mg/20 mL Type 1 clearglass vial, sealed with a 20 mm Teflon coated rubber serum stopper and atamper-evident aluminum seal. Each single use vial delivers 100 mg DrugA (5 mL) or 400 mg Drug A (20 mL) and is intended for intravenous (IV)administration.

A cycle was defined as the time from the Day 1 dose to the next Day 1dose. If there were no treatment delays, a cycle was 21 days for theweekly dosing and 28 days for the dosing every 2 weeks.

All trial treatments were administered on an outpatient basis. Patientswere observed in the clinic for at least 2 hours after infusion of DrugA on day 1 of cycle 1(C1D1) and as clinically indicated thereafter.

No premedication for Drug A was required. Guidelines in thepembrolizumab, trastuzumab and rituximab combination therapy packageinserts were followed.

In the dose escalation and expansion phases, the combination partnertherapy was administered according to its label instructions:

-   -   Trastuzumab: Initial dose of 8 mg/kg administered as an        intravenous infusion over 90 minutes, followed by 6 mg/kg        intravenous infusion (IV) administered over 30 to 90 minutes        every 3 weeks.    -   Pembrolizumab: 200 mg IV administered as an intravenous infusion        over 30 minutes every 3 weeks for up to 24 months.    -   Rituximab: 375 mg/m² administered as an intravenous infusion        once weekly for 4 doses followed by once monthly for 8 doses        (initiated at a rate of 50 mg/hr and increased by 50 mg/hr        increments every 30 minutes, to a maximum of 400 mg/hr in the        absence of infusion toxicity). If the first infusion of        rituximab was tolerated, subsequent infusions were started at a        rate of 100 mg/hr and increased by 100 mg/hr increments at        30-minute intervals, to a maximum of 400 mg/hr in the absence of        toxicity.

On administration days when dosing schedules coincided, the combinationpartner commenced approximately 30 minutes after Drug A therapyfinished. On such days, in the event of a missed dose of the Drug A drugdue to toxicity, the partner drug was administered 24 hours after themissed dose. In the event the Drug A was permanently discontinued, thepatient was discontinued from the treatment phase of the study. In theevent that the partner drug was permanently discontinued, the patientcontinued single agent Drug A for up to 24 months if in theinvestigator's opinion, the patient was deriving clinical benefit fromDrug A.

Dose Escalation Component

For Drug A, the initial dose escalation component began one dose levelbelow the single agent maximum tolerated dose (MTD) or maximumadministered dose (MAD) (3-6 patients in each dose level) taking intoaccount the observed Drug A single agent dose limiting toxicity (DLT)profile and known safety profile of the proposed combination agent(Table A). If the Drug A dose was safe and well tolerated incombination, then the dose level of Drug A was increased to the MTD orMAD with the combination agent.

No MTD for Drug A as a single agent has been reached. The MAD for Drug Aas a single agent was 30 mg/kg administered IV every other week (QOW orQ2W)

TABLE A Dose escalation for Drug A. Route of Cohort Dosage mg/kgAdministration Frequency of Dosing 1 0.3 IV Once a week 2 1.0 IV Once aweek 3 3.0 IV Once a week 4 10.0 IV Once a week 5 30.0 IV Once every 2weeks

Expansion Phase

Drug A was administered once per week (QW) at a dose of 10 mg/kg as anintravenous (IV) infusion over approximately 60 minutes on an outpatientbasis.

In the expansion phase, the combination partner therapy was administeredaccording to its label instructions, as described above.

The DrugA+pembrolizumab expansion group included up to 20 patients withmetastatic NSCLC and up to 20 patients with recurrent or metastatic headand neck squamous cell carcinoma.

The DrugA+trastuzumab expansion group included up to 20 patients withHER2-overexpressing (HER2 positive) gastric carcinoma.

The DrugA+rituximab expansion group included approximately 10 patientswith relapsed or refractory, diffuse large B cell lymphoma, andapproximately 10 patients with indolent lymphoma.

Each patient received Drug A until disease progression, unacceptabletoxicity, withdrawal of consent, or study termination. Patients receivedstudy therapy on study after radiographic progression if, in theestimation of the Investigator, the patient was deriving clinicalbenefit from the study treatment.

Efficacy Analyses

Overall response rate (ORR), disease control rate (DCR), duration ofresponse (DoR), progression free survival (PFS), and overall survival(OS) were analyzed in the ITT population and Evaluable Population.

The objective tumor response was evaluated using the using the ResponseEvaluation Criteria in Solid Tumors (RECIST) version 1.1 for solidtumors. Tumor assessments included all known or suspected disease sites.Imaging included chest, abdomen and pelvis CT or MRI scans; brain CT orMRI scan for patients with known or suspected brain metastases; bonescan and/or bone x-rays for patients with known or suspected bonemetastases. In addition, for lymphoma patients, tests included PET scansand bone marrow evaluation. The same imaging technique used tocharacterize each identified and reported lesion at baseline wasemployed in the following tumor assessments.

Antitumor activity was assessed through radiological tumor assessmentsconducted at baseline, during treatment, whenever disease progressionwas suspected (e.g., symptomatic deterioration), and at the time ofwithdrawal from the study (if not done in the previous 6 weeks).

Assessment of response was made using RECIST version 1.1 or, whererelevant, the Lugano Criteria (Cheson et al., J. Clin. Oncol (2014)32:27: 3059-3068.).

Changes in tumor size were categorized as complete response (CR),partial response (PR), stable disease (SD), or progressive disease (PD),the latter incorporating the appearance of new lesions. In the expansioncohorts, a secondary analysis was carried out using the Immune-RelatedRECIST 1.1. To facilitate this secondary analysis, in expansion cohorts,only, the diameters (longest for non-nodal lesions, shortest for nodallesions) of all target and new measurable lesions were collected.Additionally, in the expansion cohorts, confirmation of both progressionand response by imaging at least 4 weeks from the date first documentedwas required.

PK/PD and Biomarker Analyses

Drug concentrations of Drug A were measured using validated methods.Drug A serum concentrations were analyzed using a validated ligandbinding ELISA. PK parameters were determined from the respectiveconcentration-time data using standard noncompartmental methods. Actualsample collection times were used for the parameter calculations. ForDrug A, PK parameters including maximum concentration (C_(max)), time tomaximum concentration (T_(max)), area under the concentration-time curve(AUC_(last), AUC_(inf), and/or AUC_(t)) were calculated. As appropriate,additional PK parameters including clearance (CL), volume ofdistribution (Vz), terminal elimination half-life (t_(1/2)), andaccumulation ratio (R_(ac)) were calculated.

PK/PD analyses were conducted to explore the exposure-responserelationship using appropriate model-based methods to assist OBDdetermination. Pharmacodynamic data (receptor occupancy andimmunophenotyping) were summarized graphically and with descriptivestatistics by time and dose.

PK/PD analysis using appropriate model-based methods were explored tobetter understand the exposure-response relationship. Pre- and post-doselevels of CD47 target occupancy were analyzed and immunophenotyping ofcirculating leukocyte population was performed. CD47 target occupancy inperipheral blood T lymphocytes and erythrocytes was measured by flowcytometry. Infiltrating leukocyte populations and immune-modulatorymolecules in tumor biopsy tissue before and after treatment, andspecific cytokines and chemokines in serum before and after treatmentwere analyzed. Exploratory molecular analysis (including but not limitedto additional immune markers) in peripheral blood and tumor biopsysamples was performed before and after treatment.

CD8, CD68, CD163, and PD-L1 on tumor tissue were measured byimmunohistochemistry (IHC) assays. Percent positive values for CD8,CD68, and CD163 were obtained by image analysis. PD-L1 (Clone 22C3)tumor proportion score (TPS) and combined positive score (CPS) wereobtained by pathologist review. HER2 levels were determined usingHERCEPTEST™. RNA expression from paired tumor biopsies were assessedusing NANOSTRING IO360™ expression panel. Cell type abundance andpathway profiling analyses using pre-defined gene signatures wereperformed using NANOSTRING nSOLVER™ analysis software.

Blood samples were collected at the Baseline visit and retained forpharmacogenomic analyses related to drug response. For example, SIRPαgene polymorphisms, putative safety biomarkers, drug metabolizing enzymegenes, drug transport protein genes, or genes thought to be related tothe mechanism of drug action are examined.

Results

Patient Characteristics

Eighty-two patients with advanced solid tumor malignancies were enrolledin this study. The patient baseline characteristics are provided inTable B.

TABLE B Patient baseline characteristics Trastuzumab Pembrolizumab n =30 N = 52 Primary Gastric/GEJ/Esophageal 25 — Disease, n HNSCC — 20NSCLC — 26 Breast 2 — Colorectal — 2 Ovarian 1 2 Pancreatic 1 —Peritoneal — 1 Appendiceal — 1 Urothelial 1 — Median age, years (range)58 (45-79) 61 (32-81) Sex, n F 9 23 M 21 29 Race, n White 13 34 Asian 1411 Other 3 7 ECOG PS, 0 8 16 n 1 22 36

Safety

Drug A in combination with trastuzumab or pembrolizumab was welltolerated, and most treatment-related adverse events (TRAE) were of lowgrade and frequency. Treatment-related adverse events occurring in twoor more patients are provided in Table C for the Drug A+trastuzumabcombination, and in Table D for the DrugA+pembrolizumab combination. Themost frequent TRAEF in the DrugA+trastuzumab combination was fatigue(26.70%). The most frequent TRAEF the DrugA+pembrolizumab combinationwas AST increased (154).

TABLE C Treatment-related adverse events that occurred in two or morepatients treated with the Drug A + trastuzumab combination. TreatmentRelated Adverse Events Drug A + Trastuzumab (N = 30) Adverse Event Totaln (%) ≥Grade 3 Fatigue 8 (26.7) Platelets decreased 4 (13.3) 2 (6.7)Decreased appetite 3 (10)   Pyrexia 3 (10)   Anemia 2 (6.7)  Nausea 2(6.7)  Neutropenia 2 (6.7)  2 (6.7)

TABLE D Treatment-related adverse events that occurred in two or morepatients treated with the Drug A + pembrolizumab combination. TreatmentRelated Adverse Events Drug A + Pembrolizumab (N = 52) Adverse EventTotal n (%) ≥Grade 3 AST Increased  8 (15.4) ALT Increased  7 (13.5) 1(1.9) Fatigue  6 (11.5) Pruritus 5 (9.6) Anemia 4 (7.7) 1 (1.9) Infusionreaction 4 (7.7) Platelets decreased 4 (7.7) 2 (3.8) Alkalinephosphatase 3 (5.7) increased Arthralgia 3 (5.8) Pyrexia 3 (5.8) WBCdecreased 3 (5.8) Decreased appetite 2 (3.8) Myalgia 2 (3.8) Nausea 2(3.8) Neutropenia 2 (3.8) 1 (1.9) Rash 2 (3.8)

TRAEs of Grade 3 severity were of low frequency. In theDrugA+trastuzumab combination, one treatment-related serious adverseevent of febrile neutropenia was reported. In the DrugA+pembrolizumabcombination, three treatment-related serious adverse events werereported: one autoimmune hemolytic anemia, one febrile neutropenia, andone neutropenia.

Efficacy

Dose Escalation

The dose escalation component had a total of 22 patients. In theDrugA+trastuzumab combination cohort (n=10), of the 8 evaluablepatients, 3 exhibited stable disease (SD) (2 patients had breast cancer,1 patient had GEJ). In the DrugA+pembrolizumab combination cohort(n=12), of the 10 evaluable patients, 1 exhibited a partial response(PR) (NSCLC that was CPI refractory) and 3 exhibited stable disease (1patient had appendiceal cancer, 2 patients had NSCLC).

Dose Expansion

The dose expansion phase had a total of 60 patients.

DrugA+trastuzumab combination cohort with HER2 positive gastric GEJcancer (n=20): Of the 18 evaluable patients, 4 exhibited partialresponse (confirmed) and 5 exhibited stable disease. FIG. 3A providesthe percent change of disease assessment from baseline. The ORR was 22%,the DCR was 28% and the mPFS was 2.2 months. FIG. 3B provides thepercent change of disease assessment from baseline for each patient overthe study period. FIG. 3C provides the duration of treatment forenrolled patients in this cohort. Tumors were assessed according to theResponse Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (E. A.Eisenhauer, et al., European Journal of Cancer 45 (2009) 228-247.)

DrugA+pembrolizumab combination cohort with HNSCC (n=20): Of the 19evaluable patients, 3 exhibited a partial response (2 confirmed, 1unconfirmed), and 6 exhibited stable disease. FIG. 4A provides thepercent change of disease assessment from baseline. The ORR was 16% forall patients, and 30% for checkpoint therapy naïve patients; the DCR was26% for all patients, and 30% for checkpoint therapy naïve patients; andthe mPFS was 2.1 months for all patients. In addition, the combinedpositive score (CPS) for PD-L1 staining is reported for each patient.FIG. 4B provides the percent change of disease assessment from baselinefor each patient over the study period. FIG. 4C provides the duration oftreatment for enrolled patients in this cohort. Tumors were assessedaccording to the Response Evaluation Criteria in Solid Tumors (RECIST)version 1.1 (E. A. Eisenhauer, et al., European Journal of Cancer 45(2009) 228-247.)

DrugA+pembrolizumab combination cohort with NSCLC (n=20): Of the 18evaluable patients, 8 exhibited stable disease. FIG. 5A provides thepercent change of disease assessment from baseline. The disease controlrate (DCR) was 17% and the mPFS was 2 months. In addition, the tumorproportion score (TPS) for PD-L1 staining is reported for each patient.FIG. 5B provides the percent change of disease assessment from baselinefor each patient over the study period. FIG. 5C provides the duration oftreatment for enrolled patients in this cohort. Tumors were assessedaccording to the Response Evaluation Criteria in Solid Tumors (RECIST)version 1.1 (E. A. Eisenhauer, et al., European Journal of Cancer 45(2009) 228-247.)

Pharmacokinetics

Drug A PK observations from combination cohorts DrugA+trastuzumab andDrugA+pembrolizumab were within predicted 95% intervals based on anestablished population PK model (Jin F. et al., (2018) Soc Immunotherpayof Cancer Conference, #P340) (FIG. 6A). The steady-state half-life ofDrug A (10 mg/kg QW) was predicted to be approximately 16 days. As shownin FIG. 6B, near complete CD47 target occupancy on CD4+ T cells thatexpress CD47 was maintained throughout the Drug A dosing interval inboth combinations cohorts.

Anti-Tumor Response

As shown in FIG. 7A, paired biopsies from 5 NSCLC patients treated withDrugA+pembrolizumab, 6 HNSCC patients treated with DrugA+pembrolizumab,and 1 gastric cancer patient treated with DrugA+trastuzumab revealed anincrease in tumor-associated macrophages and infiltrating lymphocytesafter treatment. Images from individual patients showing staining forCD68 and CD8 before and during treatment are provided in FIG. 7B (NSCLCPD-L1(−) patient treated with DrugA+pembrolizumab) and FIG. 7C (HNSCCPD-L1(+) patient treated with DrugA+pembrolizumab). CD68 and CD163markers indicate macrophages; the CD8 marker indicates T lymphocytes.

Conclusions

Drug A in combination with pembrolizumab or trastuzumab demonstratesexcellent tolerability with favorable PK/PD characteristics. Objectiveresponses were observed in patients with late line NSCLC, HNSCC, andGastric/GEJ, including disease relapsed/refractory to prior CPI andHER2-targeted therapies.

Drug A (10 mg/kg QW; molar equivalent to 20 mg/kg of an antibody) incombination with standard regimens of trastuzumab or pembrolizumab waswell tolerated with a favorable hematologic safety profile.

Drug A demonstrates anti-cancer activity in combination with trastuzumabin HER2 positive patients that have progressed on prior HER2 targetedtherapies (e.g., HER2-positive Gastric/GEJ tumors that have progressedon prior HER2 targeted therapies).

Drug A demonstrates anti-cancer activity in combination withpembrolizumab in patients with:

-   -   ≥2L HNSCC that compares favorably to the pembrolizumab single        agent experience.    -   NSCLC, including tumors resistant/refractory to prior checkpoint        inhibitor therapy.

Drug A demonstrates antibody-like PK and complete CD47 target occupancyin combination with trastuzumab or pembrolizumab.

Preliminary data from paired tumor biopsies suggests increasedintra-tumoral macrophages and CD8+ T cells following Drug A treatment.

Taken together, the data presented in this Example demonstrate theefficacy and safety of Drug A administered in combination withpembrolizumab, trastuzumab or rituximab, in patients with advancedmalignancies including non-small cell lung cancer (NSCLC), head and necksquamous cell carcinoma (HNSCC), HER2-overexpressing gastric cancer, andnon-Hodgkin lymphoma (NHL). Without wishing to be bound by theory, asshown in FIG. 8, it is believed that Drug A, when administered incombination with anti-cancer therapeutic antibodies such aspembrolizumab, trastuzumab or rituximab, maximizes the innate andadaptive immune response to cancer. In particular, it is believed thatDrug A: 1) Enhances macrophage phagocytosis of cancer cells by blockingCD47; 2) Increases the ratio of inflammatory M tumor-associatedmacrophages (TAMs) to suppressive M2 TAMs; and 3) Activates dendriticcells (DCs) and enhances cross-priming of T cells. Furthermore, it isbelieved that Drug A in combination with anti-cancer antibodies avoidsdose-limiting toxicities associated with other CD47-target approacheswhile maximizing the innate and adaptive immune response.

Example 2A: Further Safety Results from a Phase 1 Study of Drug A inCombination with Pembrolizumab or Trastuzumab

As described in Example 1, 52 patients with solid tumor received Drug Ain combination with pembrolizumab, and 30 patients with solid tumorreceived Drug A in combination with trastuzumab. Treatment-relatedadverse effects (TRAEs) (including fatigue, AST increase, plateletdecrease, ALT increase, anemia, and/or pruritus) were of low grade andlow frequency.

35 (67.3%) of patients who received DrugA+pembrolizumab and 22 (73.3%)of patients who received DrugA+trastuzumab experienced any TRAE. Themost frequent TRAE experienced by patients who receivedDrugA+pembrolizumab was low-grade aspartate transaminase (AST) increase(17.3%), and the most frequent TRAE experienced by patients who receivedDrugA+trastuzumab was low grade fatigue (30%). TRAEs of >Grade 3severity were of low frequency. See Tables E and F below.

TABLE E TRAE in Patients Receiving Drug A + Trastuzumab Adverse EventTotal N (%) ≥Grade 3 Fatigue 9 (30)  — PLATELETS DECREASED  5 (16.7) 2(6.7) Decreased Appetite 3 (10)  — PRURITUS 3 (10)  — Pyrexia 3 (10)  —Anemia 2 (6.7) — Nausea 2 (6.7) — Neutropenia 2 (6.7) 2 (6.7)

TABLE F TRAE in Patients Receiving Drug A + Pembrolizumab Adverse EventTotal N (%) ≥Grade 3 AST Increased  9 (17.3) — ALT Increased  7 (13.5) 1(1.9) Fatigue  6 (11.5) — Anemia 5 (9.6) 1 (1.9) Pruritus 5 (9.6) — RASH5 (9.6) — Infusion Reaction 4 (7.7) — PLATELETS DECREASED 4 (7.7) 2(3.8) Alkaline Phosphatase Increased 3 (5.8) — Arthralgia 3 (5.8) —Pyrexia 3 (5.8) — WBC Decreased 3 (5.8) — Decreased Appetite 2 (3.8) —Myalgia 2 (3.8) — Nausea 2 (3.8) — Neutropenia 2 (3.8) 1 (1.9)

For Tables E and F, RASH: rash, rash papulo-macular, rash vesicular,rash pruritic dermatitis; PLATELETS DECREASED: platelets decrease,thrombocytopenia; PRURITUS: pruritus, pruritus generalized.

Four treatment-related serious adverse events (TRSAEs) were reported inpatients receiving DrugA+pembrolizumab: 1 patient experienced autoimmunehemolytic anemia/pancytopenia; 1 patient experienced febrileneutropenia; 1 patient experienced neutropenia; and 1 patientexperienced peripheral neuropathy. One TRSAE was reported in patientsreceiving DrugA+trastuzumab. The patient experienced febrileneutropenia.

Drug A displays a favorable exposure-safety relationship across theexposure ranges administered in the clinic (i.e., 10 mg/kg qw-30 mg/kgqow) with no exposure-dependent cytopenias observed.

Example 2B: Further Results from a Phase 1 Study of Drug a inCombination with Pembrolizumab in Patients with ≥2L Head and NeckSquamous Cell Carcinoma

As described in Example 1, 20 patients with ≥2L HNSCC received Drug A incombination with pembrolizumab. Baseline characteristics of all patientsreceiving DrugA+pembrolizumab (including patients with ≥2L NSCLC, asdescribed in further detail in Example 2C below) are shown in Table G.

TABLE G Baseline Characteristic of All Patients* Receiving Drug A +Pembrolizumab Drug A + Pembrolizumab N = 52 Primary Disease, n Lung 25HNSCC 20 Gastric/GEJ Esophageal — Breast — Colorectal 2 Ovarian 2Pancreatic — Appendiceal 1 Sarcoma 1 Urothelial — Unknown 1 Median AgeYears (range) 61 (32-81) Sex, n M 29 F 23 Race, n White 34 Asian 11Black 3 Other 4 ECOG PS, n 0 18 1 34 *patients included those with ≥2 LHNSCC and ≥2 L NSCLC (see Example 2C).

Anticancer efficacy in HNSCC patients was observed in response-evaluablepatients. Clinical activity was based on investigator assessed responseusing RECIST 1.1 criteria (E. A. Eisenhauer, et al., European Journal ofCancer 45 (2009) 228-247). Among the 10 patients who were checkpointinhibitor-naïve (i.e., who had not received prior treatment with animmune checkpoint inhibitor), the overall response rate (ORR) was 40%(95% CI:12.2, 73.8); the median PFS (mPFS) was 4.61 months (95% CI:0.53;7.53), and the median overall survival (mOS) had not been reached with a14.4 month median follow-up or a 17.9 month follow-up. The diseasecontrol rate (DCR) was 50% (95% CI: 18.7: 81.3): 4/10 of thecheckpoint-inhibitor naïve patients achieved partial response (“PR”) (2confirmed); 2/10 achieved stable disease (“SD”); and 4 demonstratedprogressive disease (“PD”). The duration of response (DOR) was 4.31months. Among the 10 patients who were checkpoint inhibitor-experienced(i.e., who had received prior treatment with an immune checkpointinhibitor), the ORR was 0%; the mPFS was 2.0 months [95% CI:0.9; 3.6],and the mOS as 7.4 months (95% CI: 3.1; NC). 3 patients in thecheckpoint inhibitor-experienced subgroup achieved SD, and 7demonstrated PD. See FIG. 9A, which shows the clinical activity of theDrug A+pembrolizumab combination in response-evaluable patients, andFIG. 9B, which shows the best overall response and duration of responsein ≥2L HNSCC patients treated with Drug A and pembrolizumab. Additionaldetails regarding ORR, DCR, DOR, mPFS, and mOS for all patients (i.e.,both checkpoint inhibitor experienced and checkpoint inhibitor naïve)are provided in FIG. 9A.

Full peripheral CD47 target occupancy and increased infiltrating immunecells in tumor biopsies were seen. These data confirm clinical activityof the DrugA+pembrolizumab combination treatment in patients withadvanced checkpoint inhibitor-naïve HNSCC (including PD-L1 negativepatients). The clinical activity compares favorably with historiccontrols, namely, the pembrolizumab single-agent experience. Preliminarybiomarker analyses suggested that baseline levels of CD47 and SIRPα geneexpression and tumor-infiltrating CD8⁺ cells CD68⁺ cells, and CD163⁺cells are not associated with tumor response as measured by % change oftarget lesion size from baseline.

Example 2C: Further Results from a Phase 1 Study of Drug a inCombination with Pembrolizumab in Patients with ≥2L NSCLC Who Progressedon Prior Checkpoint Inhibitor Therapy

As described in Example 1, 20 patients with ≥2L NSCLC received Drug A incombination with pembrolizumab. 17 had progressed on prior checkpointinhibitor therapy, and 3 of the patients were checkpoint-inhibitornaïve.) Baseline characteristics of all patients receiving DrugA+pembrolizumab (including those being treated for HNSCC) are shown inTable G above. Clinical activity in NSCLC patients was based oninvestigator assessed response using RECIST 1.1 criteria. The overallresponse rate (ORR) was 5% (95% CI: 0.1, 24.9), with 1 patient achievingpartial response (PR), 9 patients achieving stable disease (SD), and 10patients demonstrating progressive disease (PD). The patient whoachieved PR initially exhibited progressive disease, followed by stabledisease and subsequent partial response. The patient who achieved PR hada tumor proportion score of 0%. The disease control rate (DCR) was 35%(95% CI:15.4, 59.2). The median progression-free survival was 2.01months (95% CI: 1.88, 5.56), and the median overall survival was 9.11months (95% CI: 7.17, NC). See FIG. A, which shows the clinical activityof the Drug A+pembrolizumab combination in response-evaluable ≥2L NSCLCpatients, and FIG. 10B, which shows the best overall response andduration of response in ≥2L NSCLC patients treated with Drug A andpembrolizumab.

These data confirm clinical activity of the DrugA+pembrolizumabcombination treatment in patients with ≥2L NSCLC, including those whoare resistant/refractory to prior checkpoint inhibitor therapy.Preliminary biomarker analyses suggested that baseline levels of CD47and SIRPα gene expression and tumor-infiltrating CD8⁺ cells CD68⁺ cells,and CD163⁺ cells are not associated with tumor response as measured by %change of target lesion size from baseline.

Example 2D: Further Results from a Phase 1 Study of Drug A inCombination with Trastuzumab in Patients with ≥2L HER2-PositiveGastric/GEJ cancer

As described in Example 1, 25 patients with ≥2L HER2⁺ gastric cancer orHER2+gastroesophageal cancer received Drug A in combination withtrastuzumab. Baseline characteristics of all patients receivingDrugA+trastuzumab are shown in Table H below:

TABLE H Baseline Characteristics Drug A + Trastuzumab N = 30 PrimaryDisease, n Lung — HNSCC — Gastric/GEJ Esophageal 25 Breast 2 Colorectal— Ovarian 1 Pancreatic 1 Appendiceal — Sarcoma — Urothelial 1 Unknown —Median Age Years (range) 60 (45-79) Sex, n M 21 F 9 Race, n White 13Asian 14 Black 1 Other 2 ECOG PS, n 0 11 1 19

Clinical activity in patients with gastric cancer/GEJ cancer was basedon investigator assessed response using RECIST 1.1 criteria. Anticancerefficacy was observed in response-evaluable patients. The ORR was 21.1%(95% CI: 6.1, 45.6), the mPFS was 2.2 months (95% CI: 1.9; 5.4), the mOSwas 11.5 months (95% CI: 3.36; 14.0). Among the 19 response-evaluablepatients, 4 achieved partial response (3 confirmed); 5 achieved stabledisease; and 10 demonstrated progressive disease. The disease controlrate (DCR) was 26.3% (95% CI: 9.1, 51.2), and the duration of responsewas 9.38 months. See FIG. 11A, which shows the clinical activity of theDrug A+trastuzumab combination in response-evaluable ≥2L HER2⁺ gastriccancer or ≥2L HER2⁺ GEJ cancer patients, and FIG. 9B, which shows thebest overall response and duration of response in ≥2L HER2⁺ gastriccancer or ≥2L HER2⁺ GEJ cancer patients treated with Drug A andtrastuzumab.

Full peripheral CD47 target occupancy and increased infiltrating immunecells in tumor biopsies were seen. These data confirm clinical activityof the DrugA+trastuzumab combination treatment in patients with HER2⁺gastric or gastroesophageal cancer that have progressed on priorHER2-targeted therapies. The clinical activity compares favorably withhistoric controls. Preliminary biomarker analyses suggested thatbaseline levels of CD47 and SIRPα gene expression and tumor-infiltratingCD8⁺ cells CD68⁺ cells, and CD163⁺ cells are not associated with tumorresponse as measured by % change of target lesion size from baseline.

Example 2E: Further Results from a Phase 1 Study of Drug a inCombination with Rituximab in Patients with Non-Hodgkin Lymphoma

This example provides further results from the clinical study of DrugA's safety profile and antitumor activity in combination with rituximabwith both aggressive and indolent histologies of non-Hodgkin Lymphoma(NHL), as described in Example 1.

Patients enrolled in the study were ≥18 years of age and had relapsed orrefractory CD20-positive B-cell NIL for which no curative therapy wasavailable or were relapsed or refractory to standard approved therapies.Patients were required to have adequate organ function and hemoglobin ≥8g/dL; absolute neutrophil count ≥1,000/mm³, and platelets ≥50,000/mm³.Patients who had received prior treatment with any anti-CD47 oranti-SIRPα agent were excluded.

Patients received Drug A (10 mg/kg QW or 15 mg/kg QW) in combinationwith rituximab (375 mg/m² weekly for 4 doses followed by once monthlyfor 8 doses). The primary endpoint for the safety confirmationpopulation was first cycle dose limiting toxicity (DLT). Tumor response(using Lugano Working Group 2014 response criteria in NHL), adverseevents (characterized using NCI CTCAE v 4.03), pharmacokinetic (PK), andpharmacodynamic (PD) markers were assessed in all patients.

33 NHL patients (23 male, 10 female; median prior lines of therapy=3)were administered with Drug A in combination with rituximab. 22 patients(11 with diffuse large B-cell lymphoma (DLBCL); 4 with mantle celllymphoma (MCL); 5 with follicular lymphoma (FL); and 2 with marginalzone lymphoma (MZL)) were given Drug A 10 mg/kg QW+rituximab, and 11patients (6 with DLBCL; 1 with MCL; 3 with FL; and 1 with MZL) weregiven Drug A15 mg/kg QW+rituximab. The baseline characteristics of thepatients are shown in Table I, and patient drug exposure and dispositionare shown in Table J.

TABLE I Baseline Characteristics of Patients Receiving Drug A +Rituximab for NHL Drug A (10 mg/kg Drug A (15 mg/kg QW) + RituximabQW) + Rituximab (N = 22) (N = 11) Primary Disease, n Follicular 5 3Marginal Zone 2 1 DLBCL 11 6 Mantle Cell 4 1 Median Age Years (range) 66(32-80) 64 (53-78) Sex, n M 17 6 F 5 5 Race, n Asian 18 9 White 4 2 ECOGPS, n 0 7 2 1 15 8

TABLE J Patient Drug Exposure and Disposition Drug A + Rituximab Drug A10 mg/kg Drug A 15 mg/kg (N = 22) (N = 11) Dose Reductions, n 0 0Discontinuation Due to TRAE  1{circumflex over ( )} 0 DiscontinuationDue to PD 12  4 Discontinuation Due to Death  2* 0 Discontinuation Dueto Other 1 1 Ongoing Treatment 6 6 {circumflex over ( )}Discontinuationdue to rituximab infusion reaction; *Death due to disease progression.

No patient required a dose reduction, and the most common reason fordiscontinuation of treatment was disease progression.

Drug A in combination with rituximab was well tolerated, and mosttreatment related adverse events (TRAEs) were of low grade and lowfrequency. Twenty-six (78.8%) patients experienced any adverse event.Fifteen (45.5%) patients experienced any TRAE. The most common TRAE ofDrug A in combination with rituximab was Grade 1-2 rash (18%), fatigue(9%, n=3), anemia (6%, n=2), nausea (6%, n=2), and neutropenia (6%,n=2). TRAEs ≥Grade 3 severity were of low frequency (see Table K). Notreatment related serious adverse events were reported. There were 2deaths on study, both due to disease progression.

TABLE K Treatment-Related Adverse Events Adverse Event Total N (%)≥Grade 3 Rash  6 (18%) — Fatigue 3 (9%) — Nausea 2 (6%) — NeutrophilCount Decreased 2 (6%) 2 (6%) Anemia 2 (6%) 1 (3%)

No Drug A dose limiting toxicities were reported. The maximum tolerateddose (MTD) of Drug A in combination with rituximab was not reached. Themaximum administered dose of Drug A (i.e., administered in combinationwith rituximab) was 15 mg/kg QW. No significant exposure-cytopeniarelationship was observed across the Drug A exposure range evaluated (10mg/kg QW-15 mg/kg QW).

Anti-tumor activity was observed across all histologies inresponse-evaluable patients with relapsed/refractory aggressivehistologies (i.e., DLBCL and MCL) and relapsed/refractory indolenthistologies (i.e., FL and MZL). Responses were evaluated according toLugano 2014 response criteria (see Cheson et al. (2014) “Recommendationsfor Initial Evaluation, Staging and Response Assessment of Hodgkin andNon-Hodgkin Lymphoma: The Lugano Classification.” J. Clin Oncol. 32:3059-3067). As shown in Table L and FIG. 12, the overall response rate(ORR) among patients treated with 10 mg/kg QW DrugA+rituximab was 40.9%.3 Patients (i.e., one with Mantle Cell Lymphoma (MCL), one withFollicular Lymphoma (FL), and one with MZL) achieved complete response(CR), 6 patients (two with Diffuse Large B-Cell Lymphoma (DLBCL), twowith MCL, and two with FL) achieved partial response (PR). 6 patients(two with DLBCL, one with MCL, two with FL, and one with MZL)demonstrated stable disease (SD). The ORR among patients treated with 15mg/kg QW DrugA+rituximab was 54.6% ORR. 2 patients (both with FL)achieved CR. 4 patients (one with MZL, two with DLBCL, and one with MCL)achieved PR. 1 patient (with FL) demonstrated SD. See Table L below andFIG. 12. Further details regarding the duration of treatment forpatients administered with 10 mg/kg DrugA+rituximab are provided in FIG.13A, and further details regarding the duration of treatment forpatients administered with 15 mg/kg DrugA+rituximab are provide in FIG.13B.

TABLE L Drug A + Rituximab Combination - Clinical Activity inResponse-Evaluable Patients Median Median Median Median Population ORRDOR PFS OS Follow-Up Drug A Dose N (95% CI) (95% CI) (95% CI) (95% CI)(95% CI) NHL (10 mg/ 22 40.9% NC 7.4 NC 11.3 kg ALL) (23.3; 61.3) (1.9;13.2) (10.2; 15.2) NHL (10 mg/ 15 33.3% 5.6 2.5 8.9 10.4 kg aggressive)(15.2; 58.3) (1.8; NC) (1.0; 7.4) (2.5; NC) (9.5; 15.2) NHL (10 mg/ 757.1% NC NC NC 11.3 kg indolent) (25.1; 84.2) (7.8; 20.4) NHL (15 mg/ 1154.6% NC NC NC 5.1 kg ALL) (28.0; 78.7) (4.0; 5.6) NHL (15 mg/ 7 42.9%NC 1.9 NC 4.7 kg aggressive) (15.8; 75.0) (1.1; NC) (4.0; 5.4) NHL (15mg/ 4 75% NC NC NC 5.9 kg indolent) (30.1; 95.4) (3.5; 7.4) Aggressive =relapsed refractory DLBCL or relapsed refractory mantle cell lymphoma;Indolent = relapsed refractory follicular lymphoma or relapsedrefractory marginal zone lymphoma; ORR = objective response rate(complete response + partial response); mDOR = median duration ofresponse (months); mPFS = median progression free survival (months);mFollow-Up = median follow up (months); NC = could not be calculated.

Across the exposure range evaluated (10 mg/kg QW-15 mg/kg QW), increasedDrug A exposure was observed in subjects with a best response of CR andPR compared to subjects with a best response of SD and PD. FavorableDrug A pharmacokinetics and CD47 receptor occupancy were seen across thedosing interval.

Drug A in combination with standard regimens of rituximab was welltolerated with a favorable hematologic safety profile and no maximumtolerated dose reached. The maximum administered dose was 15 mg/kg QW(molar equivalent to 30 mg/kg QW of an antibody) with no exposuredependent anemia, thrombocytopenia or neutropenia observed across theexposure range evaluated. Drug A demonstrates emerging anti-canceractivity with durable responses in combination with rituximab inpatients with relapsed/refractory NHL whose tumors have progressed onprior CD20 targeted therapies that compares favorably to historiccontrols. Preliminary data suggests Drug A is well tolerated and thathigher exposure of Drug A is observed in responders vs non-responders.

Each embodiment herein described may be combined with any otherembodiment or embodiments unless clearly indicated to the contrary. Inparticular, any feature or embodiment indicated as being preferred oradvantageous may be combined with any other feature or features orembodiment or embodiments indicated as being preferred or advantageous,unless clearly indicated to the contrary.

All references cited in this application are expressly incorporated byreference herein.

1. A method of treating non-small cell lung cancer (NSCLC) in anindividual, comprising administering to the individual an effectiveamount of (a) a polypeptide comprising a SIRPα D1 domain variant and anFc domain variant, and (b) an anti-PD-1 antibody wherein the SIRPα D1domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and deG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, deG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the individual is a human.
 2. The method of claim 1, wherein theNSCLC in the individual has progressed on a prior checkpoint inhibitor(CPI) therapy and/or has a PD-L1 tumor proportion score (TPS) of lessthan 50%,
 3. (canceled)
 4. A method of treating head and neck squamouscell carcinoma (HNSCC) in an individual, comprising administering to theindividual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-PD-1antibody, wherein the SIRPα D1 domain variant comprises the amino acidsequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domainvariant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A,and N297A mutations, wherein numbering is according to the EU index ofKabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297Amutations, wherein numbering is according to the EU index of Kabat;(iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, anddelG236 mutations, wherein numbering is according to the EU index ofKabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, delG236, and N297A mutations, wherein numbering is according tothe EU index of Kabat; and wherein the HNSCC in the individual hasprogressed while on a prior platinum therapy or after the prior platinumtherapy, and wherein the individual is a human.
 5. The method of claim4, wherein the individual has received prior therapy with an immunecheckpoint inhibitor.
 6. The method of claim 4, wherein the individualhas not received prior therapy with an immune checkpoint inhibitor. 7.The method of claim 4, wherein the prior platinum therapy comprised oneor more therapeutic agents selected from the group consisting of:cisplatin, carboplatin, and oxaliplatin.
 8. The method of claim 1,wherein the anti-PD-1 antibody is pembrolizumab.
 9. (canceled)
 10. Amethod of treating HER2-positive gastric/gastroesophageal junction (GEJ)cancer in an individual, comprising administering to the individual aneffective amount of (a) a polypeptide comprising a SIRPα D1 domainvariant and an Fc domain variant, and (b) an anti-HER2 antibody, whereinthe SIRPα D1 domain variant comprises the amino acid sequence of SEQ IDNO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a humanIgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations,wherein numbering is according to the EU index of Kabat; (ii) a humanIgG2 Fc region comprising A330S, P331S, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (iii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations,wherein numbering is according to the EU index of Kabat; or (iv) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the gastric/GEJ cancer in the individual has progressedfollowing a prior treatment with a fluoropyrimidine-based therapy and/ora prior treatment with an anti-HER2 antibody, and wherein the individualis a human.
 11. (canceled)
 12. The method of claim 10, wherein theanti-HER2 antibody is trastuzumab.
 13. (canceled)
 14. A method oftreating aggressive non-Hodgkin lymphoma (NHL) in an individual,comprising administering to the individual an effective amount of (a) apolypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant, and (b) an anti-CD20 antibody, wherein the SIRPα D1 domainvariant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the aggressive NHL in the individual is relapsed and/orrefractory to a prior treatment for aggressive NHL and there is noavailable curative therapy, and wherein the individual is a human. 15.The method of claim 14, wherein the aggressive NHL is a de novo diffuselarge B-cell lymphoma (DLBCL) or a transformed DLBCL.
 16. (canceled) 17.The method of claim 14, wherein the aggressive NHL is a mantle celllymphoma (MCL).
 18. (canceled)
 19. A method of treating indolentlymphoma in an individual, comprising administering to the individual aneffective amount of (a) a polypeptide comprising a SIRPα D1 domainvariant and an Fc domain variant, and (b) an anti-CD20 antibody, whereinthe SIRPα D1 domain variant comprises the amino acid sequence of SEQ IDNO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a humanIgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations,wherein numbering is according to the EU index of Kabat; (ii) a humanIgG2 Fc region comprising A330S, P331S, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (iii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations,wherein numbering is according to the EU index of Kabat; or (iv) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the indolent lymphoma in the individual is relapsed and/orrefractory to a prior treatment for indolent lymphoma, and wherein theindividual is a human.
 20. The method of claim 19, wherein the indolentlymphoma is an indolent non-Hodgkin lymphoma (NHL).
 21. The method ofclaim 20, wherein the indolent NHL is a marginal zone lymphoma or afollicular lymphoma.
 22. (canceled)
 23. The method of claim 14, whereinthe anti-CD20 antibody is rituximab.
 24. (canceled)
 25. The method ofclaim 1, wherein the SIRPα D1 domain variant comprises the amino acidsequence of SEQ ID NO:
 85. 26. The method of claim 1, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NOL: 81.27. The method of claim 1, wherein the Fc domain variant is a human IgGFc region comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat.
 28. The method of claim27, wherein the Fc domain variant comprises the amino acid sequence ofSEQ ID NO:
 91. 29. The method of claim 1, wherein the polypeptidecomprising a SIRPα D1 domain variant and an Fc domain variant comprisesthe amino acid sequence of SEQ ID NO:
 136. 30. The method of claim 1,wherein the polypeptide comprising a SIRPα D1 domain variant and an Fcdomain variant comprises the amino acid sequence of SEQ ID NO:
 135. 31.The method of claim 1, wherein the polypeptide comprising a SIRPα D1domain variant and an Fc domain variant forms a homodimer.
 32. Themethod of claim 1, wherein the polypeptide comprising a SIRPα D1 domainvariant and an Fc domain variant is administered to the individual at adose of 10 mg/kg once per week (QW).
 33. The method of claim 14, whereinthe polypeptide comprising a SIRPα D1 domain variant and an Fc domainvariant is administered to the individual at a dose of 15 mg/kg once perweek (QW).
 34. (canceled)
 35. A kit comprising a polypeptide comprisinga SIRPα D1 domain variant and an Fc domain variant in a pharmaceuticallyacceptable carrier, for use in combination with pembrolizumab fortreating non-small cell lung cancer (NSCLC) in an individual in needthereof, wherein the SIRPα D1 domain variant comprises the amino acidsequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domainvariant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A,and N297A mutations, wherein numbering is according to the EU index ofKabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297Amutations, wherein numbering is according to the EU index of Kabat;(iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, anddelG236 mutations, wherein numbering is according to the EU index ofKabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V,L235A, delG236, and N297A mutations, wherein numbering is according tothe EU index of Kabat; and wherein the individual is human. 36.(canceled)
 37. A kit comprising a polypeptide comprising a SIRPα D1domain variant and an Fc domain variant in a pharmaceutically acceptablecarrier for use in combination with pembrolizumab for treating head andneck squamous cell carcinoma (HNSCC) in an individual in need thereof,wherein the SIRPα D1 domain variant comprises the amino acid sequence ofSEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) ahuman IgG1 Fc region comprising L234A, L235A, G237A, and N297Amutations, wherein numbering is according to the EU index of Kabat; (ii)a human IgG2 Fc region comprising A330S, P331S, and N297A mutations,wherein numbering is according to the EU index of Kabat; (iii) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236mutations, wherein numbering is according to the EU index of Kabat; or(iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations, wherein numbering is according to the EUindex of Kabat; and wherein the HNSCC in the individual has progressedwhile on a prior platinum therapy or after the platinum therapy, andwherein the individual is human.
 38. (canceled)
 39. A kit comprising apolypeptide comprising a SIRPα D1 domain variant and an Fe domainvariant in a pharmaceutically acceptable carrier for use in combinationwith trastuzumab for treating HER2-positive gastric/gastroesophagealjunction (GEJ) cancer in an individual in need thereof, wherein theSIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1Fc region comprising L234A, L235A, G237A, and N297A mutations, whereinnumbering is according to the EU index of Kabat; (ii) a human IgG2 Fcregion comprising A330S, P331S, and N297A mutations, wherein numberingis according to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the HER2-positive gastric/GEJ cancer in the individual hasprogressed following a prior fluoropyrimidine-based therapy or a priortreatment with an anti-HER2 antibody, and wherein the individual ishuman.
 40. (canceled)
 41. A kit comprising a polypeptide comprising aSIRPα D1 domain variant and an Fc domain variant in a pharmaceuticallyacceptable carrier for use in combination with rituximab for treatingaggressive non-Hodgkin lymphoma (NHL) in an individual in need thereof,wherein the SIRPα D1 domain variant comprises the amino acid sequence ofSEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) ahuman IgG1 Fc region comprising L234A, L235A, G237A, and N297Amutations, wherein numbering is according to the EU index of Kabat; (ii)a human IgG2 Fc region comprising A330S, P331S, and N297A mutations,wherein numbering is according to the EU index of Kabat; (iii) a humanIgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236mutations, wherein numbering is according to the EU index of Kabat; or(iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations, wherein numbering is according to the EUindex of Kabat; and wherein the aggressive NHL in the individual isrelapsed and/or refractory to a prior treatment for aggressive NHL andthere is no available curative therapy, and wherein the individual ishuman. 42-44. (canceled)
 45. A kit comprising a polypeptide comprising aSIRPα D1 domain variant and an Fc domain variant in a pharmaceuticallyacceptable carrier, for use in combination with rituximab for treatingindolent lymphoma in an individual in need thereof, wherein the SIRPα D1domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations, wherein numberingis according to the EU index of Kabat; (ii) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations, wherein numbering isaccording to the EU index of Kabat; (iii) a human IgG4 Fc regioncomprising S228P, E233P, F234V, L235A, and delG236 mutations, whereinnumbering is according to the EU index of Kabat; or (iv) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, delG236, and N297Amutations, wherein numbering is according to the EU index of Kabat; andwherein the indolent lymphoma in the individual is relapsed orrefractory to a prior treatment for indolent lymphoma, and wherein theindividual is human. 46-57. (canceled)